Inactivation of T Antigen-Forming Capacities of Simian Virus 40 and Adenovirus 12 by Ultraviolet Irradiation

Methods to measure T antigen-forming capacities of simian virus 40 (SV40) and adenovirus 12 (Ad12) were investigated, and a method to measure the capacity in terms of T antigen-forming units was employed by the use of cytosine arabinoside. Plaque-forming units and T antigen-forming units of SV40, SV40 deoxyribonucleic acid, or Ad12 were inactivated by ultraviolet (UV) irradiation at the same rate, roughly following a single-hit curve. T-antigen formation by UV-irradiated SV40 and Ad12 was enhanced in cells multiply infected and in cells in a growing state. These observations showed that it was difficult or impossible to estimate the size of the gene for T antigen by UV inactivation.     

Replication of Deoxyribonucleic Acid in Escherichia coli C Mutants Temperature Sensitive in the Initiation of Chromosome Replication

An Escherichia coli HF4704S mutant temperature sensitive in deoxyribonucleic acid (DNA) synthesis and different from any previously characterized mutant was isolated. The mutated gene in this strain was designated dnaH. The mutant could grow normally at 27 C but not at 43 C, and DNA synthesis continued for an hour at a decreasing rate and then ceased. After temperature shift-up, the increased amount of DNA was 40 to 50%. When the culture was incubated at 43 C for 70 min and then transferred to 27 C, DNA synthesis resumed after about 50 min, initiating synchronously at a fixed region on the bacterial chromosome. The initiation step in DNA replication sensitive to 30 mug of chloramphenicol per ml occurs synchronously before the resumption of DNA replication after the temperature shift-down, being completed about 30 min before the start of DNA replication. When the cells incubated at 27 C in the presence of 30 mug of chloramphenicol per ml after the temperature shift-down to 27 C were transferred to 43 C with simultaneous removal of the antibiotic, no resumption of DNA replication was observed. When the culture was returned to 43 C after being released from high-temperature inhibition at 30 min before the start of DNA replication, no recovery replication was observed; whereas at 20 min, the recovery of replication was observed. These results indicated that HF4704S was temperature sensitive in the initiation of DNA replication. Analysis of HF4704S, by an interrupted conjugation experiment, indicated that gene dnaH was located at about 64 min on the E. coli C linkage map. In E. coli S1814 (a K-12 derivative), which was a dnaH(ts) transductant from HF4704S (C strain) with phage P1, the mutated gene (dnaH) was demonstrated to be closely linked to the thyA marker by conjugation and P1 transduction experiments and to be distinct from genes dnaA through dnaG.     

Direct fluorimetric measurement on the hydrolysis of β-naphthyl triphosphate, a fluorescent ATP analog, by heavy meromyosin

A fluorescent ATP analog, β-naphthyl triphosphate, was hydrolyzed to β-naphthyl diphosphate and orthophosphate by heavy meromyosin ATPase. In the process of hydrolysis the fluorescence intensity of β-naphthyl triphosphate changed remarkably. Thus, the rate of β-naphthyl triphosphate hydrolysis is evaluated directly and continuously by measuring the time course of fluorescence intensity.In the presence of Ca²⁺, the Michaelis constant (K_m) of β-naphthyl triphosphate hydrolysis by heavy meromyosin was similar to that of ATP hydrolysis. While, in the presence of Mg²⁺ the K_m of β-napthyl triphosphate hydrolysis was 9.0·10⁻⁶ M, much larger than the value of ATP hydrolysis, indicating that the apparent affinity of the enzyme for β-naphthyl triphosphate is less than that for ATP.The pH dependence of β-naphthyl triphosphatase activity resembled that of ATPase activity, suggesting a similarity in the mechanism of hydrolysis of the two substrates.     

Glucose catabolism during aging and differentiation in hypocotyls ofPhaseolus mungo seedlings

Changes in activities of the glycolytic and pentose phosphate (PP) pathways in glucose catabolism in various parts of the hypocotyls obtained from 4-day-old etiolatedPhaseolus mungo seedlings were investigated by measuring the inhibition rates of respiration by iodoacetate and malonate, and the release of¹⁴CO₂ from [1-¹⁴C]- and [6-¹⁴C]glucose. The relative activity of the PP pathway in glucose catabolism was higher in the immature part (Part I) and the aged part (Part V) of the hypocotyls than in the intermediary one (Part III), while the activity of the glycolytic pathway decreased with aging. On a fresh weight basis, the enzyme activities of the glycolytic and PP pathways were higher in Part I than in Parts III and V. On a protein content basis, however, activities of the enzymes of the PP pathway increased with aging and differentiation of the hypocotyls whereas those of the glycolytic pathway decreased. Levels of nicotinamide adenine nucleotides were found to be in the following order: Part I>Part III> Part V for NAD⁺+NADH; Part I>Part V>Part III for NADP⁺+NADPH. The stimulative effect of methylene blue on decreasing the C₆/C₁ ratio was greater in Part III than in Part I, and No effect was observed in Part V. These data suggest that a decrease in the activity of the glycolytic pathway with aging and differentiation may be due to the decreasing glycolytic enzyme activities and NAD(H) content. The higher activity of the PP pathway in the immature part is attributable to larger amounts of NADP(H) and enzymes of the PP pathway. The greater contribution of the PP pathway to glucose catabolism in the aged part than in the intermediary part seems to results from a more active turnover of NADP and the relatively higher activity of the enzymes of the PP pathway than those of the glycolytic pathway.     

Performance of a perforated plate column as a multistage continuous fermentor

Microorganisms were continuously cultivated in multistage column consisting of ten perforated plate sections to which medium and air were supplied concurrently from the bottom. At steady state the cell concentration in the various stages was gradationally differentiated from the bottom to the top in the direction of medium flow. RNA content per unit cell concentration at each sage was determined. The cells in the lower stages were higher in RNA content than those from the upper stages. Wash out was observed to occur in the column at dilution rates which do not result in wash out in a single stage chemostat system. A study of the flow characteristics revealed that the overall performance of the plate column was equivalent to that of a multistage system, when hole diameter and hole area to column cross sectional area ratio were properly selected. This was true even in highly aerated conditions. These results indicated that the perforated plates in the column hindred intermixing through the plates, and that each stage functioned as an independent stirred vessel. Industrial and research application of this type fermentor was discussed.     

Solution properties of synthetic polypeptides. V. Helix–coil transition in poly(β-benzyl L-aspartate)

Simple approximate expressions have been derived from the theory of Zimm and Bragg for use in the analysis of experimental data on the helix-coil transition in polypeptide. On the basis of the resulting expressions practical procedures are proposed to determine two basic parameters characterizing a thermally induced transition, i.e., helix initiation parameter σ and enthalpy change for helix formation, ΔH. They have been applied to the data for poly(β-benzyl L -aspartate) (PBLA) with the result: σ = 1.6 × 10^?4 and ΔH = ?450 cal/mole for PBLA in m-cresol; σ = 0.6 × 10^?4 and ΔH = 260 cal/mole for PBLA in chloroform containing 5.7 vol-% of dichloroacetic acid. This result gives evidence that σ may change not only from one polypeptide to another but also for a given polypeptide in different solvents. The change in limiting viscosity number [η] accompanying the transition was measured in the same solvents. The curve of [η] versus helical content had a relatively monotonic shape for the chloroformdichloroacetic acid solutions as compared with that for the m-cresol solutions, indicating that [η] depended largely on σ. Provided that [η] is a direct measure of the mean-square radius of gyration, 〈S²〉, the results are consistent with the theoretical predictions of Nagai and of Miller and Flory for 〈S²〉.