Thomas W. James

Assay for nanogram quantities of DNA in cellular homogenates

Abstract The DNA concentration of a crude cellular homogenate can be measured accurately in the nanogram range using the fluorescence enhancement of 4′,6-diamidino-2-phenylindole (DAPI) or bisbenzimidazole (Hoechst H 33258) complexed with DNA. A simple assay has been devised including an internal standard, which allows reliable measurement and compensates for any quenching due to cellular components or buffer. The fluorescence enhancement is highly specific for DNA; no other cell component produces significant fluorescence. The response is linear over a broad dynamic range making the measurement of unknown DNA concentrations convenient.

Synchronization of cell division in Astasia longa on a chemically defined medium

Abstract 1. 1. A 24-hour repeating temperature cycle, consisting of 15 hours at 15 °C, 8 hours at 25 °C, and two 30-minute transition periods, has been used for the synchronization of cell division of Astasia longa grown in 2 per cent proteose peptone and in a chemically defined medium. This results in a burst of cell division every 24 hours for as many as seven generations. 2. 2. Cysteine and methionine were found to be important in the mechanism of synchronization of cells grown in Cramer-Myers medium. Their addition appears to reduce the fission time of these cells without affecting the generation time. 3. 3. The synchronization process is evaluated in terms of a cell cycle. The possible manner in which synchronization is achieved by means of a repeating temperature cycle is discussed.

Light-induced division synchrony in Euglena gracilis var. Bacillaris

Abstract Reserve energy of the photosynthetic flagellate Euglena gracilis when grown in continuous light of saturating intensity was estimated by following growth after removal of the culture to darkness. This information permitted the construction of a light-dark cycle, the light period of which provided energy just sufficient for growth and division for one life cycle, the temperature being maintained constant at 20 °C. Divisions were synchronous and repetitive, the population essentially doubling in number at each division “burst”. Addition of cysteine and methionine to the basal salt medium reduced the variance of the division bursts.

The effect of incubation temperature on the cell size of Tetrahymena pyriformis

Abstract The growth of Tetrahymena pyriformis at three different incubation temperatures, 10 °, 20 ° and 30 °C, results in three classes of cell size having average volumes of 16,250 μ 3 , 12,350 μ 3 , and 9375 μ 3 respectively. The frequency distributions of each class were determined, and they indicate that the differences in the averages are not merely the result of weighting the distribution with any one cell volume since there is a shift in the entire range of the volume distributions. The Q O 2 (10 6 cells) were also measured. The larger cells have the higher rate of oxygen consumption when measured at 20 °C, having Q O 2 (10 6 cells) of 66, 38 and 23 respectively. The surface to volume ratios were determined as this ratio was considered to be one factor which may govern the change in cell size with temperature. The ratios tend to remain constant. The size change cannot be entirely attributed to surface-dependent metabolism.

The role of the nucleus in the maintenance of ribonucleic acid in Amoeba proteus

Abstract The RNA content of whole amoebae, nucleated half amoebae, and enucleated half amoebae has been followed as a function of time after the cells are cut and placed in a balanced salt solution. The curves for the loss of RNA by the three cell types are used to calculate initial rates of loss for each type. An RNA replacement rate is calculated for the nucleus of both the nucleated half amoebae and the intact amoebae. The two rates agree reasonably well.

Parametric analysis of volume distributions of Schizosaccharomyces pombe and other cells

Abstract Time-lapse photomicrographic data have been obtained on mating strains of the yeast Schizosaccharomyces pombe to evaluate the effect of the variability of the patterns of cell cycle behavior on population structure. These have been used to design a computer model which accepts volume distribution data from exponential cultures of a cell and yields estimates of the mean and standard deviation of daughter cell volume and telophase cell volume, as well as a stop-grow point, and the degree of cell volume doubling. Given a cell populations volume distribution and a volume distribution from a subpopulation, the program will estimate the mean age and display how the age is distributed in the subpopulation. Several cell types have been examined.

Oxygen binding characteristics of lobster hemocyanin and its subunits

Abstract 1. 1. Dissociation of lobster hemocyanin was broguht about by changes in pH. The effect of dissociation and reassociation of subunits on the oxygen equilibrium characteristics of hemocyanin was studied. Dissociation was determioned by ultracentrifugation and electrophoresis. Oxygen dissociation curves were obtained using a tonometer equipped with both a polarographic sensor and a 1 -cm quartz cuvette. 2. 2. The hemocyanin molecule remained aggregated between pH 4·5 and pH 8·5. Subuniting occured between both pH 3·0–4·5 and pH 8·5–10·5. The subunits sedimented with values of 8·5 S and 6·5 S , while the aggretated form of the molecule and an average value of 16·0 S . 3. 4. The reassociation of subunits was not complete, and although the autocatalytic nature of the ligand-binding reaction was not chagned in reassociated hemocyanin, the total amount of oxygen bound per molecule was slightly less. 4. 5. The numner of ligand-binding sites per molecule, as indicated by Hill equation analysis, is at least four.

The configuration of adenosine triphosphate as deduced from rotatory dispersion.

Abstract The rotary dispersion of adenine, adenosine, AMP, ADP and ATP have been determined. The data have been interpreted to show that the ATP molecule is folded back upon itself in such a manner that bonding is permitted between the last two phosphate groups and the amino group of adenine. This stabilized structure is then proposed as necessary for the action of ATP. Parallel “ATP-like” action of CTP and UTP and other triphosphonucleotides could be explained by possession of a similar configuration.

Rough endoplasmic reticulum-mitochondrial complexes from rat liver. An extramitochondrial succinic dehydrogenase associated with this rough endoplasmic reticulum.

Abstract The objective of this investigation was to determine if a mitochondrial inner membrane protein coded for by the nuclear genome could be identified with the rough endoplasmic reticulum associated with mitochondria. Rough endoplasmic reticulum-mitochondrial complexes were collected by zonal centrifugation. These associations were then subjected to hypotonic-hypertonic treatment and the mitoplasts were separated from rough endoplasmic reticulum and outer membranes. These were analysed for various marker enzymes including succinic dehydrogenase, succinate cytochrome c reductase, and cytochrome oxidase for inner mitochondrial membrane, monoamine oxidase for outer mitochondrial membrane, and glucose-6-phosphatase for rough endoplasmic reticulum. Some inner membrane damage occurred and contaminated the RER fraction as determined by cytochrome oxidase activity. However, the ratios of monoamine oxidase: cytochrome oxidase and glucose-6-phosphatase: cytochrome oxidase were over 20-fold and 105-fold greater, respectively, than for intact mitochondria indicating that inner mitochondrial membrane contamination was very low. Interestingly, succinic dehydrogenase activity was found to be present in the rough endoplasmic reticulum-outer mitochondrial membrane fraction, but succinate cytochrome c reductase activity was not detectable. In addition, the ratio of succinic dehydrogenase: cytochrome oxidase activity was 9-fold higher in the RER fraction than in mitochondria. These results were interpreted to mean that the succinic dehydrogenase activity in this fraction did not represent an enzyme which was a component of the electron transport chain. Specific activity of succinic dehydrogenase in the rough endoplasmic reticulum-outer mitochondrial membrane fraction was diminished in material from fasted rats and was increased in fasted-refed animals. Succinic dehydrogenase was also found localized on the rough endoplasmic reticulum by electron microscopic procedures using tetrazolium salts. These experiments support the hypothesis that succinic dehydrogenase is synthesized on the mitochondrially associated RER in preparation for transfer to the mitochondria.

Detection of A + T-rich DNA in gels by differential fluorescence

The fluorochrome Hoechst 33258 preferentially forms complexes with A + T-rich duplex DNA, whereas ethidium bromide binds nucleic acids independent of base composition. Both compounds can be conveniently used to visualize DNA fractionated by gel electrophoresis. Determination of fluorescence emission from Hoechst 33258-stained restriction fragments normalized to fluorescence derived from the same sample after ethidium bromide staining provides a measure of emission due to A + T content, and allows easy identification of A + T-rich restriction fragments. To demonstrate the utility of this procedure, an A + T map of bacteriophage lambda DNA was constructed and found to be comparable to similar maps derived by alternate techniques. Analysis of recombinant plasmid DNAs with established nucleotide sequences demonstrated that the A + T content of individual restriction fragments could be estimated to within an accuracy of 5%.