Dean B. Cowie

Metabolic pools and the synthesis of macromolecules

Abstract Two functionally distinct amino acid pools are foundin Candida utilis : a concentrating pool and a conversion pool . The former accumulates exogenous amino acids within the cell to levels exceeding their concentration in the external medium. This pool is only evident when exogenous amino acids are present. Its size is variable and the accumulated amino acids are sensitive to osmotic shock as well as being readily exchangeable with external amino acids. Once concentrated these amino acids may provide material for the conversion pool. Here amino acid interconversion occurs thereby furnishing the amino acids required for protein synthesis. In the absence of exogenous amino acids this pool is formed solely from the carbon and energy source (glucose, fructose, etc.). During exponential grwoth this pool is always present at a fixed size. This amino acid conversion pool is sensitive to osmotic shock. Its content do not exchange with the amino acids of the concentrating pool or with exogenousamino acids. The results are similar to those obtained in the synthesis of nucleic acids in Candida utilis where two functionally different and chemically distinct pools have been demonstrated. These kinetic investigations describing the flow of oxegenous carbon through metabolic pools provide information on some of the preliminary steps in macromolecule formation.

Amino acid analog incorporation into bacterial proteins

The amino acid analogs norleucine and para-fluorophenylalanine are shown to be incorporated into the proteins of E. coli. Analysis of proteins by an ion-exchange column showed that the proteins formed in the presence of the analogs are not radically different molecular species but are physicochemically similar to the proteins normally synthesized. The substitution of norleucine for methionine in the bacterial proteins was shown to occur in the same proportions in all of the “protein classes” resolved by the ion-exchange column.

Kinetics of formation and utilization of metabolic pools in the biosynthesis of protein and nucleic acid

A study of the kinetics of formation and utilization of metabolic pools of amino acids and phosphorus in T. utilis has been carried out using 14C-fructose and 32PO4---. The distribution of these tracers among the chemical fractions of the cell during steady-state conditions was first established. The interrelationships among these fractions in the formation of metabolic pools and in the transfer of pool material for protein and nucleic acid synthesis were then determined. The pools were demonstrated to be complex and so the kinetics of formation and transfer of individual pool amino acids were investigated. These investigations and a study of the effects of wxogenous amino acid competitors upon pool amino acids led us to the following conclusions. 1. 1. The carbon of the cold TCA-soluble fraction is mostly contained in amino acids and acts as a metabolic pool for the synthesis of protein and nucleic acid. The TCA-soluble fraction also contains a metabolic pool of phosphorus which supplies phosphorus for nucleic acid and other end products found in the hot TCA-soluble fraction. 2. 2. The results of kinetic investigations permit the distinction between endogenous metabolic pools as essential intermediates for synthesis and reservoir pools not on the main line of synthetic events. The amino acid and phosphorus pools are in the direct line of synthetic events converting exogenous substrate to endogenous macromolecular compounds. 3. 3. The pool amino acids are not free amino acids but are adsorbed to larger molecules and these adsorption sites are intimately connected with the process of protein synthesis. 4. 4. Internal conversion of one pool amino acid to another pool amino acid may occur on a single site prior to incorporation into protein. 5. 5. Nucleic acids are probably not the macromolecules furnishing the adsorption sites since there are more than 3 times as many pool amino acids as there are nucleotides. The protein fraction is the only cellular component present in sufficiently large quantities to furnish the adsorption sites for the pool amino acids. A mixture of nucleic acid and protein is not excluded however.

The use of metabolic pools of purine compounds for nucleic acid synthesis in yeast

Abstract Exogenous purines are incorporated first into metabolic pools and thence into nucleic acids by exponentially growing Candida utilis. Kinetic studies show that a purine pool, whose size is determined by the amount of purine in the medium, forms first. This pool contributes material to a nucleotide pool whose pool size is independent of the purine concentration in the medium. Conversion of adenine to guanine occurs in the nucleotide pool which serves as a preferred source of purine for nucleic acid synthesis.

Metabolic pools and the utilization of amino acid analogs for protein synthesis

Abstract Studies of the kinetics of analog and amino acid incorporation into metabolic pools and proteins of yeast and E. coli have shown that at least two processes are involved in the selection of the natural amino acid in preference to the analog. These selection steps occur in the formation of the metabolic pools, the first occurring in the concentrating system of the cell, which accumulates exogenous amino acids or analogs into the cell at levels exceeding external concentrations. In the yeast, the final distinction between analog and amino acid occurs at the time of entry into a second metabolic pool (internal pool). In this pool conversion of amino acids to the required end products occurs. Once incorporated into this pool no further selection occurs; the ratio of analog to amino acid in this pool and in protein is the same.

DNA homology among the T-even bacteriophages

Abstract DNA-DNA hybridization studies involving reactions between labeled T-even phage DNA fragments and excess unlabeled T-even phage DNAs gave results which indicated that (a) as expected, close genetic similarities among the T2-T4 and T6 DNAs exist; (b) because reciprocal experiments (labeled T2 DNA reacted with T4 DNA and labeled T4 DNA reacted with T2 DNA, etc.) gave unequal values, different molecular weights are predicted for each of the three phage DNAs. Relative to the molecular weight of T2 DNA the molecular weights of T6 and T4 are 8 and 10% larger, respectively; (c) the differences of the thermal stabilities of the hybrid duplexes denote a closer genetic relationship between the T2 and T6 DNAs than to the T2 and T4 or T4 and T6 DNAs.

The labeling of bacterial cells with S35 for the production of high specific activity compounds.

Summary These results demonstrate that the 23.4 mc. of bound sulfur (Table I) taken up by 0.15 ml. of cells provide a means of specifically labeling both methionine and cystine (2×10 9 counts/sec./mg. of sulfur) without interference from excessive radiation exposure of the cells. It is also apparent that extremely small numbers of E. coli cells can be labeled and detected.

Arc Ion Source with Direct‐Current Filament Supply for 60‐Inch Cyclotron

A CAPILLARY arc ion source for a 60‐inch cyclotron is described utilizing a direct‐current filament supply. This has permitted 80‐hour filament life with large deuteron beams. Stability of the arc and its operating conditions and construction are indicated.

Genetic interrelationships among the T-even bacteriophages

Abstract Genetic interrelationships among the T-even bacteriophages have been studied by DNA-DNA hybridization techniques. The isolation, identification and characterization of completely distinct nucleotide sequences from heteroduplexes provided DNA fractions which, upon further testing, gave results useful for a comparative evaluation of the viral contents of the T-even bacteriophages.