Studies are also being conducted by Pollard and associates at Pennsylvania State University in an attempt to formulate a theoretical basis for the description of the processes of synthesis, growth, division, and differentiation of the living cell. Such a theory would be basic to an understanding of very primitive life forms or prebiological material which might be found elsewhere in the universe. For these purposes, studies are being undertaken in macromolecular reproduction which differ from the studies involving cellular genetic material. Theories concerning the problem of replication of cellular structures and information storage in two-dimensional systems are being developed. Theories are also being developed about the mechanisms which control and regulate receptor and enzymatic activities within the cell.
One study involved the rate of mutation in cells and disposed of the suggestion that the process of mutation consists of a "tunneling" of proton from one base to another in DNA. Such a suggestion can no longer be advanced as a major explanation of mutations.
Work is also being conducted on the centrifugation of cells of E. coli. It has been shown that cells exposed to as little as 100 g have a modification in their function. This has been looked at from the point of view of thymine uptake, which would be concerned with the formation of DNA, and also from the point of view of the induction of an enzyme, which would correspond to the transcription of the DNA. Preliminary experiments in the latter case indicate considerable centrifugation effect. The thymine uptake is affected, but not nearly as much as formerly thought. Further work is in progress in this area.
Important work has been completed on the cells of E. coli grown on maltose, which can be induced to produce betagalactosidase by the addition of thiomethyl galactoside. 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. This enables the calculation of the half-life of unstable messenger RNA. The half-life for this decay is readily measurable, and values are given over a temperature range of 17° C (5.2 minimum) to 45° C (0.56 minimum). These agree very well with half-lives measured by others by inducing for short times and measuring the course of enzyme formation. The rate of transcription is involved in the kinetics of cessation of enzyme induction, and the rate of transcription can be measured. Arrhenius plots for this rate and the rate of decay are given, and the activation energies measured are about 16 000 cal/mole. The cessation of transcription is linked to the degradation, possibly of only one strand, of DNA.
Pollard has suggested that one important action of ionizing radiation is concerned with the transcription of the genetic message into RNA. Clayton and Adler ([ref.159]) showed that induced catalase synthesis in Rhodopseudomonas spheroides is inhibited by low doses of X-rays, giving experimental support to the idea. Pollard and Vogler ([ref.160]), using cells in which the process of induction involved permease, showed that there is some sensitivity to gamma radiation. Novelli et al. ([ref.161]) found a reduced sensitivity as compared with colony formation, but it is still a considerable sensitivity.
The process of induction of an enzyme indicates that the transcription of the genetic message is repressed by something which can be acted on by a small molecule, the inducer, to remove repression and permit the formation of messenger RNA, which then acts to make the enzyme. The messenger RNA undergoes decay through a process which is still not clear. Very elegant measurements by Kepes ([ref.162]) show that for the messenger RNA for betagalactosidase, the half-life is 1.02 min at 37° C and 2.05 min at 25° C. The time of onset of enzyme formation after induction was found to be about 3 minutes.
If the process of transcription is indeed sensitive to ionizing radiation, then the irradiation of cells which have just been induced should show formation of the enzyme to the extent of formation of new messenger RNA within a few minutes, plus the formation of the enzyme while the messenger RNA is decaying. This pattern was found by Clayton and Adler. The experiments conducted by Pollard and associates amplify and extend their work and also agree with the work of Kepes ([ref.162]).
BIOINSTRUMENTATION
Fernandez-Moran (refs. [ref.163]-[ref.165]), at the University of Chicago, has devised a new multielectrode electrostatic lens which he has incorporated into an electron microscope. This necessitated the development of a novel high-voltage power source and voltage regulator of extreme stability and accuracy. Some promising work has now been done on superconducting lenses. In a series of experiments with a simple electron microscope without pole pieces, using high-field superconducting niobium-zirconium solenoid lenses in an open air core, liquid helium Dewar, electron microscopic images of test specimens have been recorded while operating at 32 200 gauss in a persistent current mode, with regulated accelerating potentials of 4 to 8 kilovolts. These preliminary experiments have demonstrated the exceptional stability of the images (both short term and long term) over a period of 4 to 8 hours and the relatively high quality of the images.
Progress has been made on the viscosimeter for high intrinsic viscosities. This is now working, and the viscosity of DNA preparations has been measured. It is hoped to use the viscosimeter to study the variation in DNA viscosity as a function of the cell cycle.