Janice M. Nigro

Human p53 and CDC2Hs genes combine to inhibit the proliferation of Saccharomyces cerevisiae.

Human wild-type and mutant p53 genes were expressed under the control of a galactose-inducible promoter in Saccharomyces cerevisiae. The growth rate of the yeast was reduced in cells expressing wild-type p53, whereas cells transformed with mutant p53 genes derived from human tumors were less affected. Coexpression of the normal p53 protein with the human cell cycle-regulated protein kinase CDC2Hs resulted in much more pronounced growth inhibition that for p53 alone. Cells expressing p53 and CDC2Hs were partially arrested in G1, as determined by morphological analysis and flow cytometry. p53 was phosphorylated when expressed in the yeast, but differences in phosphorylation did not explain the growth inhibition attributable to coexpression of p53 and CDC2Hs. These results suggest that wild-type p53 has a growth-inhibitory activity in S. cerevisiae similar to that observed in mammalian cells and suggests that this yeast may provide a useful model for defining the pathways through which p53 acts.

Efficient singlet oxygen inactivation of firefly luciferase

Firefly luciferase is inactivated by singlet oxygen at near diffusion controlled rates, 1.9 X 10(9) M-1 s-1, based on direct comparison with the oxidation of L-histidine. The inactivation kinetics are multiphasic. Inactivation is inhibitable by NaN3. Surface-separated-sensitizer (SSS) system in which singlet oxygen is produced above an air gap separating the reaction solution from the Rose Bengal sensitizer, ensuring only Type II reactions, was compared with a Sensitox II system in which the polymer bound Rose Bengal is contained in the reaction solution and both Type I and Type II reactions can occur. A slight stabilization is afforded by MgSO4.