Yeast bcy1 mutants with stationary phase-specific defects.

Entry into the stationary phase requires the yeast BCY1 gene, which encodes the regulatory subunit of the cAMP-dependent protein kinase (cAPK). New bcy1 mutants, constructed by in vitro mutagenesis of the 3'-region encoding the cAMP-binding domains, were classified as early or late-acting mutants based on viability studies. The late-acting bcy1 mutants accumulated fewer stationary phase-specific Bcy1p isoforms and had decreased cAPK activity. This late-acting class is novel and dies after 7 days in culture, later than two previously reported stationary phase mutants, ubi4 and ard1.

Stationary phase in Saccharomyces cerevisiae.

Like other microorganisms, the yeast Saccharomyces cerevisiae responds to starvation by arresting growth and entering stationary phase. Because most microorganisms exist under conditions of nutrient limitation, the ability to tolerate starvation is critical for survival. Molecular analyses have identified changes in transcription, translation, and protein modification in stationary-phase cells. At the level of translation, the pattern of newly synthesized proteins in stationary-phase cells is surprisingly similar to the pattern of proteins synthesized during exponential growth. When limited for different nutrients, yeast strains may not enter stationary phase but opt for pathways such as pseudohyphal growth. If nutrient limitation continues, the end-point is likely to be a stationary-phase cell. Based on the results of recent studies, we propose a model for entry into stationary phase in which G(o) arrest is separable from acquisition of the ability to survive long periods of time without added nutrients.

A DNA polymerase alpha-associated 56 kDa protein kinase.

Initiation of chromosomal DNA replication must be carefully regulated during the cell cycle. We report that Drosophila embryo DNA polymerase alpha complex, isolated by immunological techniques, contains a protein kinase activity. The kinase will phosphorylate histone H1, but prefers peptides contained in the DNA polymerase alpha-kinase complex. Renaturation experiments determined that the kinase activity resides in a 56-kDa peptide.

Delta-type DNA polymerase characterized from Drosophila melanogaster embryos.

Genetic and biochemical evidence suggests there are at least three DNA polymerases required for replication in eukaryotic cells. However, Drosophila embryonic cells have a very short duration S phase which is regulated differently. To address the question of whether embryos utilize different DNA polymerases, we employed Mono Q anion exchange chromatography to resolve the DNA polymerase activities. Two types of DNA polymerase, DNA polymerase delta and DNA polymerase alpha, were distinguished by: 1. copurification of DNA primase or 3'-5'exonuclease activities; 2. immunoblot analysis with alpha-specific polyclonal antisera; 3. sensitivity to aphidicolin and BuPdGTP; and 4. processivity measurements with and without Proliferating Cell Nuclear Antigen. These observations suggest that Drosophila embryos, similar to nonembryonic cells, have both alpha- and delta-type DNA polymerases.

Toxic effects of acute glutathione depletion by buthionine sulfoximine and dimethylfumarate on murine mammary carcinoma cells.

Glutathione (GSH) depletion to approximately equal to 5% of control for 48 h or longer by 0.05 mM L-buthionine sulfoximine (BSO) led to appreciable toxicity for the 66 murine mammary carcinoma cells growing in vitro [L.A. Dethlefsen et al., Int. J. Radiat. Oncol. Biol. Phys. 12, 1157-1160 (1986)]. Such toxicity in normal, proliferating cells in vivo would be undesirable. Thus the toxic effects after acute GSH depletion to approximately equal to 5% of control by BSO plus dimethylfumarate (DMF) were evaluated in these same 66 cells to determine if this anti-proliferative effect could be minimized. Two hours of 0.025 mM DMF reduced GSH to 45% of control, while 6 h of 0.05 mM BSO reduced it to 16%. However, BSO (6 h) plus DMF (2 h) and BSO (24 h) plus DMF (2 h) reduced GSH to 4 and 2%, respectively. The incorporation (15-min pulses) of radioactive precursors into protein and RNA were unaffected by these treatment protocols. In contrast, cell growth was only modestly affected, but the incorporation of [3H]thymidine into DNA was reduced to 64% of control by the BSO (24 h) plus DMF (2 h) protocol even though it was unaffected by the BSO (6 h) plus DMF (2 h) treatment. The cellular plating efficiencies from both protocols were reduced to approximately equal to 75% of control cells. However, the aerobic radiation response, as measured by cell survival, was not modified at doses of either 4.0 or 8.0 Gy. The growth rates of treated cultures, after drug removal, quickly returned to control rates and the resynthesis of GSH in cells from both protocols was also rapid. The GSH levels after either protocol were slightly above control by 12 h after drug removal, dramatically over control (approximately equal to 200%) by 24 h, and back to normal by 48 h. Thus even a relatively short treatment with BSO and DMF resulting in a GSH depletion to 2-5% of control had a marked effect on DNA synthesis and plating efficiency and a modest effect on cellular growth. One cannot rule out a direct effect of the drugs, but presumably the antiproliferative effects are due to a depletion of nuclear GSH with the subsequent inhibition of the GSH/glutaredoxin-mediated conversion of ribonucleotides to deoxyribonucleotides. However, even after extended treatment, upon drug removal, GSH was rapidly resynthesized and cellular DNA synthesis and growth quickly resumed.

Endogenous thiol levels in heterogeneous murine tumor cells as a function of the physiological state and the response to X-irradiation.

The endogenous thiols (PSH, protein sulfhydryls; NPSH, nonprotein sulfhydryls; and GSH, glutathione) were measured in the 66 and 67 murine carcinoma cells growing under different physiological conditions in vitro (e.g., proliferation, P; nutrient-deprived quiescence QI; and QI cells stimulated by refeeding the monolayer in situ and assayed 4 (St4) and 14 (St14) h later). The aerobic radiation response was also studied as a function of the physiological state and thiol concentration. The changes in PSH levels suggest that the proportion of thiol-containing proteins changed whenever the cells were in transition between different physiological states (e.g., when QI cells were stimulated by refeeding, the proportion of PSH was elevated dramatically over either QI or P cells). The NPSH and GSH levels were both down significantly in the QI vs. P cells as was the total thiol level (PSH plus NPSH). Fourteen h but not 4 h after stimulation, the NPSH and GSH levels had returned to or exceeded the P-cell levels. Also, the proportion of GSH in the NPSH fraction varied as a function of the physiological state. The 66 and 67 QI cells were both more radiosensitive than the respective P cells. Also, the 66 cell radiation-induced cytotoxicity had returned to the P response by about 4 h after refeeding but the stimulated 67 cells had not. However, no overall correlation was apparent between the various aerobic radiation responses and the pool sizes of either the total thiols or of the various subsets of thiols. The depressed total thiol level and the increased radiosensitivity of the QI cells could represent a cause-and-effect relationship or these parameters could be independent phenomena only related indirectly through the reduced metabolic activity of the quiescent cells.

Toxic effects of extended glutathione depletion by buthionine sulfoximine on murine mammary carcinoma cells.

Extended depletion of glutathione to approximately equal to 5% of control in the murine mammary carcinoma cell line 66 was achieved with a concentration of 0.05 mM buthionine sulfoximine. At 24 hours, there was no evidence for cellular toxicity from the BSO treatment per se; however, by 48 hours, there was inhibition of protein and DNA synthesis and cell growth and cell kinetic data was suggestive of both a G1 and a G2 block. Glutathione depletion to this extent (i.e., 0.13 mM vs. 2.24 mM in control) did not modify the aerobic radiation response for cells in the physiological states of proliferation, quiescence, or stimulated quiescent cells. This degree of cellular toxicity may well be cell-type dependent, but the results do suggest that caution is in order if one should attempt long-term GSH depletion in vivo.