Ernest C. Pollard

Theoretical studies on living systems in the absence of mechanical stress

Abstract Small single cells should show little effect of weightlessness. Cells as large as 10 μ across should show some effect due to the redistribution of mitochondria or the nucleolus. It is probable that if there is such a thing as a “gravity receptor” in a cell, it is the nucleolus. Systems of cells in which relatively large objects, such as ribosomes, pass from cell to cell should show gravity effects. Quite extensive systems, such as plants which exceed 1 cm in length should have increased membrane permeability at the lower end. This effect could be greatly increased if each cell had many internal membranes. Experiments on weightlessness should also consider temperature as a variable.

The effect of hydrostatic pressure on the synthetic processes in bacteria

Abstract An effect of hydrostatic pressure on the induction of an enzyme and on the incorporation of thymine (DNA synthesis), of proline and valine (protein synthesis) and, to a lesser extent, of uracil (RNA synthesis) has been observed. The cells are not markedly reduced in volume at the pressure used (about 900 atm). Some effect of pressure in newly synthesized protein, operating within a minute or two of synthesis, is observed.

Collision kinetics applied to phage synthesis, messenger RNA, and glucose metabolism☆

Abstract Elementary collision kinetics is applied to the processes of phage DNA and protein synthesis, messenger RNA combination with ribosomes and glucose metabolism. Except for the case of messenger RNA and ribosomes, the process of collision, with reasonable diffusion constants, is adequately rapid to explain the observed rates. The formation of a temporary complex, which eliminates the requirement of perfect collisions, is shown to be unlikely to slow diffusion very greatly. The case of collision where the numbers are too small to allow the use of the diffusion equation is examined, and it is seen that cells with dimensions in excess of one micron will require some mechanism to guarantee collision. The presence of an activation energy will require that successive reactions be produced in an organized system. It is suggested that the in vitro synthesis of a bacterial cell should be possible if small reaction vessels can be designed.

RADIATION ACTION ON DNA IN BACTERIA: EFFECT OF OXYGEN.

In Escherichia coli cells ionizing radiation produces a degradation of DNA to approximately 50 percent of the total amount originally insoluble in trichloroacetic acid. There is also a reduction of synthesis. Oxygen inhibits the degradation process by a dose reduction factor of 4 and the synthesis process by a factor of approximately 1.5. Thus, at relatively low doses, radiation action on bacteria is very probably mediated through the DNA.

IONIZING RADIATION: EFFECT ON GENETIC TRANSCRIPTION

Cells of Escherichia coli grown on maltose can be induced by the addition of thiomethyl galactoside to produce β-galactosidase. If cells are irradiated shortly after induction, the transcription of the DNA ceases, and the enzyme produced by the messenger RNA is observed to reach a maximum. From these data the calculated half-life of unstable messenger RNA is given over a temperature range from 8.1 minutes at 10�C to 0.7 minute at 45�C. The kinetics of cessation of transcription give information on both meassenger RNA decay and rate of transcription. Arrhenius graphs for both these rates are given, and the activation energies mtieasured are 11,000 calories per mole for decay and 22,000 calories per mole for transcription. This relation to temperature is characteristic of enzymatic behavior.

Physical aspects of protein and DNA synthesis

Abstract The idea that the synthetic processes of a bacterial cell take place by random collision and specific selection has been examined in terms of Brownian movement theory. The observed rates of protein, deoxyribonucleic acid and ribosome synthesis are used to calculate the necessary concentrations of the metabolites used in the synthetic process. If the collision process must involve the presentation of the active part of the colliding molecule at first impact, then the observed rates require excessive concentrations for soluble ribonucleic acid. The concentration required for nucleotide triphosphates in the synthesis of deoxyribonucleic acid is such that many points of synthesis are called for. If, instead, a very temporary complex can form, of the nature expected from the operation of London-Van der Waals forces, the free rotation of the colliding molecule provides a variety of aspect changes, and the collision process is much more efficient. If this process takes place, the transfer of energy from rotation to vibration becomes important. The precision of assembly of deoxyribonucleic acid is examined in terms of the idea that precision in timing is important. It is shown that the cell could not function in that way if any large molecule had to move during the assembly. It is suggested that enzymes having high activation energies must be present in abnormally high concentrations.

Relation to ultraviolet light mutagenesis to a radiation-damage inducible system in Escherichia coli.

Revertants produced by ultraviolet light (uv) were shown to follow a square-law dependence on fluence up to about 0.5 J/m/sup 2/, after which the dependence became closer to linear. This behavior can be associated with an induction process, and a new way of expressing mutation data is presented that allows one to estimate the fraction of cells in a population that is inducible after exposure to a particular uv fluence. Comparison with two other kinds of radiation-induced behavior (induction of inhibition of post-irradiation DNA degradation and induced radioresistance) shows that the fluence dependence of induction is similar in all three cases. A predose with ionizing radiation followed by an incubation period and subsequent exposure to a graded set of uv fluences at 265 nm gave an increase in the number of revertants and a more linear dose dependence. The same result was obtained if the predose was a uv fluence at 265 nm and the graded set of doses was at 313 nm, a wavelength which by itself produces very few revertants. These changes in the mutagenic response were also consistent with the induction of a mutation-potentiating system by the predose. Ultraviolet-produced mutations in the presence or absence of anmorexa0» ionizing radiation predose appear to be due to C ..-->.. T changes in the bacterial genome (C and T denote cytosine and thymine, respectively).«xa0less

Ionizing Radiation: Effect of Irradiated Medium on Synthetic Processes

The incorporation of uracil-C14 into macromolecules in Escherichia coli cells is decreased by doses of ionizing radiation when the cells are in very dilute suspension. The decrease results from an action of irradiated medium on the cells, and a similar reaction is observed during the incorporation of thymine (indication of DNA synthesis) and of proline and valine (indicative of protein synthesis). Irradiated medium reduces the formation of β-galactosidase but does not cause the degradation of DNA.

An analog computer study of differential equations concerned with bacterial cell synthesis

Abstract A set of seven differential equations has been proposed to describe the synthetic processes of the bacterial cell. The analog computer proved to be a suitable tool for solving the equations and for examining them in detail under a variety of circumstances. Five sets of constants from an array of possible solutions were obtained for both linear and cyclic DNA syntheses. The over-all range of these constants was not great. The solutions to the cellular equations were unique in the sense that when the decay constants were specified, the remaining constants were determined. By varying successively several different constants in one set pertaining to the linear synthesis of DNA, a variety of cellular conditions were observed. A stress was applied to the model cell by means of changing the initial conditions of its components and this effect was examined over several generations. One important result of this test was that greater stability was obtained for fast read out and fast decay than for slow read out and slow decay. Another significant result was that the greatest instability was found for three stresses applied simultaneously. Of the two different synthetic patterns for DNA, this method showed that DNA synthesized during only part of a cycle is considerably more stable. The constants relating the syntheses of structural RNA and structural ribosomes appeared to be rigidly determined over the range of solutions tested. The method of stability in successive generations was also applied to alterations of these constants. It was found that quite small changes do indeed affect stability and that this stress is more readily tolerated if both constants are altered in the same direction. The model cell responded to the condition of a magnesium deficiency in very much the same way as bacterial cells under an actual experimental test. The analog computer method proves to be quite powerful in describing the nature of a system such as the bacterial cell, even in a very simplified form.

NEAR ULTRAVIOLET INACTIVATION STUDIES ONESCHERICHIA COLI TRYPTOPHANASE AND TRYPTOPHAN SYNTHETASE

Abstract— –The response of two pyridoxal‐phosphate‐requiring enzymes of E. coli, tryptophanase and tryptophan synthetase, to near UV light (320–400 nm) has been studied. Tryptophanase is inactivated both in vivo and in vitro, but tryptophan synthetase is resistant to near UV under both conditions. This shows that near UV inactivation is not general for pyridoxal‐phosphate‐requiring enzymes. Substrate protection against light inactivation is demonstrated for tryptophanase. It is furthermore shown that pyridoxal phosphate is required for inactivation of this enzyme. However, the action spectrum for this inactivation does not coincide with the absorption spectrum of tryptophanase or of pyridoxal phosphate.