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W R Guild

Publications and source records attributed to W R Guild.

At least 37 records · Page 2Linked to original sources
PubMed1978

Transfection in pneumococcus: single-strand intermediates in the formation of infective centers.

Transfection has been found and characterized in pneumococcus. For replicating omega3 phage DNA extracted from infected cells, transfection was relatively efficient and rose linearly with DNA concentration and quadratically with time, according to T(T - 3.5) min(2). For mature DNA extracted from phage particles, transfection was hardly detectable below 1 mug/ml but increased about as the cube of the DNA concentration up to 100 mug/ml, and was still rising at concentrations over 200 mug/ml. The kinetics suggest a dependence on a mixed cubic function of the time of exposure of cells to mature DNA. Cell and phage DNAs competed with each other for transformation and transfection. Transfection was reduced much more strongly than transformation in cells that were deficient in the membrane-bound endonuclease required for conversion of donor duplex DNA to intracellular single strands; these data agree with the kinetic data in implying that independent entry of segments of two strands is necessary for transfection by replicating omega3 phage DNA and entry of at least three strands is necessary for transfection by mature DNA. To reconcile differing DNA concentration dependences of transfection and transformation with a common entry path, it was necessary to reexamine data on transformation and to recognize that this process continued to rise slowly through the concentration region usually described as "plateau." These results and the transfection data reflect multiple binding and nicking events that occurred on the cell surface before entry. Our conclusion is that transfection in pneumococcus occurs by association inside the cell of segments of single strands of phage DNA that have entered independently, creating gapped structures that need repair synthesis to create infective centers. Physical recombination is therefore automatically a prerequisite to transfection.

Bacteriophages↗
PubMed1976

Mismatch correction in pneumococcal transformation: donor length and hex-dependent marker efficiency.

A hypothesis that preferential rejection of donor markers by the hex system of pneumococcus is due to lethal double-strand breaks has been examined in terms of its implications for the extent of the excision required. Experiments reported here were directed at asking whether hex-dependent marker efficiency depends on the length of the donor deoxyribonucleic acid (DNA). In the absence of intracellular competition for hex function, there was no detectable effect of DNA size on hex-dependent marker efficiency as donor DNA was sheared from greater than 1 x 107 daltons to 3.6 x 105 daltons. The latter DNA was purified by two successive velocity fractionations to ensure that the activity seen was representative of DNA of that size. Quantitative examination of the system shows that, for the lethal event hypothesis to be true, the excision step has to remove an average of 7,000 to 10,000 nucleotides. This figure is so much greater than that seen in other excision processes that alternate hypotheses should be considered. The presently known properties of the hex system can be accounted for by a model invoking the migratory features of type I restriction enzymes.

DNA Repair↗
PubMed1976

Characterization of some pneumococcal bacteriophages.

The growth of pneumococcal phages at high cell and phage densities is enhanced strongly by the substitution of potassium for sodium in the medium. Initial titers of 2 X 10(10) to 4 X 10(10) PFU/ml are readily obtained, and concentrated stocks are stable in a storage buffer described here. The mechanism of the cation effect is obscure. Phages omega3 and omega8 each have linear double-stranded DNA of 33 X 10(6) daltons per particle, with an apparent guanine plus cytosine content of 47 to 49 mol%, as determined by buoyancy and melting temperature, but with an unusual absorbance spectrum. Efficiency of plating is high if sufficient time is allowed for a relatively slow adsorption, which differs several-fold in rate between the two phages. Morphologically, these and other pneumococcal phages are similar to coliphage lambda but with a longer tail and tail fiber. Upon UV inactivation, omega3 and omega8 have D37 values of 33 and 55 J/m2, respectively, and each shows multiplicity reactivation. A total of 13 ts mutants have been isolated from the two phages, representing only two complementation groups; complementation and recombination occur between omega3 and omega8 mutants. Both phages provoke high-titer antisera with extensive cross-reactivity against a number of newly isolated pneumococcal phages.

Adsorption↗
PubMed1973

Structure of deoxyribonucleic acid on the cell surface during uptake by pneumococcus.

We exposed competent cells of Diplococcus pneumoniae to high-molecular-weight donor deoxyribonucleate (DNA) and examined the state of the DNA bound to them in forms sensitive to deoxyribonuclease I. The portion elutable with 5 M guanidine hydrochloride was shown to be native, of much lower molecular weight (4 x 10(6) to 5 x 10(6)) than the donor, and as active in further transformation as sheared DNA of the same size. The portion resistant to release by guanidine hydrochloride was also shown to be native and active in transformation. These results, along with previous ones, imply that the breaks produced outside the cell are not at genetically specific sites. Furthermore, it was found that entry past the cell barrier to deoxyribonuclease could occur at 0 C by a process sensitive to ethylenediaminetetraacetate.

Carbon Isotopes↗
PubMed1972

Kinetics of integration of transforming DNA in pneumococcus.

Integration of donor genes, as measured by recovery of their transforming activity from eclipse in lysates of newly transformed cells of pneumococcus, has been followed at temperatures from 0 to 40 degrees . There is a lag in the recovery curve that is marker-dependent and increases as temperature falls. An Arrhenius plot of the rates shows a sharp break between 15 and 20 degrees . Brief exposure of the system to 37 degrees before incubation at 10 or 15 degrees removes the lag and raises the subsequent rate of recovery. This activation is unstable, however, and disappears when the cells are held at 0 degrees after the exposure at 37 degrees and before incubation at 15 degrees . The results are interpreted in terms of a reaction sequence A right harpoon over left harpoon B --> C, with activation energies for the first forward rate-constant of the order of 50 Cal/mol, for the second, 20-21 Cal/mol, and for the reverse reaction, less than 20 Cal/mol. The properties of the first step, including its marker dependence, are the same as those observed earlier for stabilization of donor markers against intracellular inactivation, and it is suggested they may reflect an activation of the recipient chromosome prerequisite to synapsis.

Bacteriolysis↗
PubMed1972

Events occurring near the time of synapsis during transformation in Diplococcus pneumoniae.

A marker-specific and strongly temperature-dependent reaction was observed to occur at a time during transformation in Diplococcus pneumoniae after the donor deoxyribonucleic acid (DNA) had acquired single-strand properties and immediately preceding the integration of these strands into the recipient chromosome. Operationally, it was observed as the prevention of an intracellular inactivation process, also described in this paper, which is specific for low molecular weight or for damaged DNA, and which occurs if the recipient cells are held at suboptimal temperatures after the DNA has entered. Brief exposure of the cells to a higher temperature stabilized the DNA against this inactivation, in a two step process. It is the first step which has a strong temperature dependence (DeltaHdouble dagger = 70 kcal/mole, DeltaSdouble dagger = 160 entropy units), is marker specific, and which appears to be reversible. The second step is much less temperature-dependent and overlaps in time the start of integration. The enthalpy and entropy of activation are both consistent with those needed to open a loop of six to eight base pairs in a DNA duplex. It is suggested that these observations may reflect, and provide an assay for, the kinetics of synapsis, which on this model is limited in rate by the appearance of unpaired regions on the recipient duplex.

Chromosomes, Bacterial↗
PubMed1972

Activity of deoxyribonucleic acid fragments of defined size in Bacillus subtilis transformation.

The transforming activity of Bacillus subtilis deoxyribonucleic acid (DNA) that had been sheared and purified with respect to size by sucrose gradient sedimentation is given as a function of the DNA molecular weight. It is shown (i) that fragments of median molecular weight 1.2 x 10(6) have finite activity (10(-4)), (ii) that the shape of the activity-versus-molecular weight function is qualitatively similar to that observed previously for Diplococcus pneumoniae, and (iii) that this shape precludes interpretation in terms of critical size models.

Bacillus subtilis↗
PubMed1972

Transformation and deoxyribonucleic acid size: extent of degradation on entry varies with size of donor.

The fate of label introduced as donor deoxyribonucleic acid (DNA) into competent cells of Diplococcus pneumoniae was determined immediately after entry at 25 C, as a function of the size of the donor DNA. Part of the label is found to be acid soluble, part has been incorporated into chromosomal DNA, apparently through reincorporation of degraded donor DNA, and part is found in single strands of length smaller than that of the input donor DNA strands. The last fraction apparently constitutes the precursor for integration of intact donor genetic markers and is referred to as the intact fraction. For large donor DNA the intact fraction contains over 80% of the total intracellular label, but the median strand length has been reduced to 2.2 x 10(6) daltons. For small donor molecules (1 x 10(5) to 6 x 10(5) daltons per strand) the fraction intact increases with donor size from 10 to 50% of the total intracellular label, and the median strand length of this fraction is half that of the donor strands. By combining these results with earlier data on the size dependence of the yield of transformants per unit of total intracellular donor label, we have calculated the probability that a marker in the intact fraction will be integrated, as a function of the length of the donor strand after entry. This probability has a linear dependence on strand length for activities below 40% of maximum, and extrapolates to zero activity at 77,000 daltons per strand.

Bacillus subtilis↗
PubMed1970

Competent Diplococcus pneumoniae accept both single- and double-stranded deoxyribonucleic acid.

The transforming activity of fractionated complementary strands of Diplococcus pneumoniae deoxyribonucleic acid (DNA) bands at the position of fully denatured DNA in CsCl at pH 11.0, and is completely (> 99.8%) destroyed by digestion with exonuclease-I. These results prove that pure single strands transform the normally prepared competent cells of this species. Their efficiency is about 0.5% that of native DNA of comparable size.

Cesium↗
PubMed1969

Number of transformable units per cell in Diplococcus pneumoniae.

Analysis of frequencies of single and random multiple transformations in Diplococcus pneumoniae showed that there are at least two transformable units per cell of the total population in highly competent cultures. If 100% of the cells are competent in these cases, the units may be interpreted as the strands of one duplex deoxyribonucleic acid recipient chromosome. The theory is developed to allow for extension to more complex situations.

Chromosomes, Bacterial↗