Density gradient procedures for the selection of synchronous cells of Synechococcus lividus and Chlorella sorokiniana, and the application of the isopycnic technique to the study of the patterns of phosphoribosylglycinamide synthetase during the cell cycle of Chlorella
Sitz, Thomas Orr
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The blue-green alga, Synechococcus lividus, was isolated free from contaminating bacteria by selecting colonies off agar plates which had been maintained in an atmosphere of 4% CO₂-air. Attempts were made to synchronize the algae by intermittent illumination, isopycnic centrifugation, and differential centrifugation. Cells selected by differential centrifugation were the only ones to give acceptable synchrony with cell number increasing in a short division time and a high cross-wall index (85%). Because the density of the green alga Chlorella sorokiniana appears to increase during the cell cycle, synchronous cultures of this organism were successfully selected with isopycnic centrifugation. The enzyme phosphoribosylglycinamide synthetase was found to be stable in Chlorella cells frozen and thawed once, provided substrates were present in the buffer. When frozen-thawed cells or fresh cells were broken in the French press or by a sonic oscillator the enzyme was no longer dependent on substrates for stability. However, cells broken by sonication showed an ATP-MgCl₂ dependent linear loss of enzyme activity on incubation at 38.5°. Breaking fresh cells twice in the French press in the presence of substrates, yielded an enzyme preparation with the highest activity and stability. This enzyme increased in a continuous exponential fashion, paralleling the increase in total protein, during the cell cycle of cells selected by the isopycnic procedure. Because the enzyme continued to increase at the same exponential rate during the period of DNA replication, the increase in gene dosage did not appear to affect the increase in activity of the enzyme during the cell cycle.
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