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We showed that the heat killing ,curve for exponentially growing Saccharomyces cerevisiae was biphasic. This suggests two populations of cells with different therpial killing characteristics. Whep exponentially growing cells separated into cell cycle-specific fractions via centrifugal elutriation were, heat shocked, the fractions enriched in smnall unbudded cells showed greater resistance to heat killing than did other cell cycle fractions. The lethal effects of extreme temperature on Saccharomyces cerevisiae have been described. Exponentially growing cells are much more sensitive than are stationary-phase cells to a thermal shock (15). Thus, resting or quiescent cells are more thermotolerant than growing cells are. This heat resistance is acquired as the cells pass from the exponentially growing to the stationary state (12). Schenberg-Frascino and Moustacchi (15) isolated small unbudded cells from stationary cultures and observed their thermotolerance as they progressed through the first cell cycle into exponential growth. They observed that thermotolerance is decreased in budding cells. Yeast cells, when starved for an essential nutrient such as nitrogen, phosphorous, or sulfur, cease dividing and arrest their cell division as unbudded cells. The thermotolerance of cells starved for any of these nutrients resembles that of stationary-phase cells, and it has been suggested that this thermotolerance is a general characteristic of resting cells (11).Our aim was to clarify the relationship between the cell cycle and thermotolerance. To this end we examined the effect of the cell cycle position on the thermotolerance of S. cerevisiae in exponentially growing cultures by using the elutriator rotor to separate cells by size and cell cycle position (9). We also examined the effects on the cell cycle of a mild heat shock, during which the cells acquire thermotolerance (10, 13). In addition, we used several methods to arrest cells in the unbudded state, and we determined the thermotolerance of the resulting populations. The methods included arrest via deprivation of essential nutrients, alphafactor arrest, and the use of temperature-sensitive cell division cycle mutants. Our results demonstrate that a distinct population of unbudded cells was more thermotolerant than were cells in other morphological stages in the cell cycle. We suggest that this thermotolerance is associated with a defined state, G0, which has synthetic and physiological properties distinct from those of other unbudded cells. MATERIALS AND METHODSStrains. The following strains were used: SKQ2n (a/a adell+ +/ade2 +Ihisl); A364A (a lys2 tyrl his7 gall adel ade2 ural); cdc7-1 (ts124); cdc25-1 (ts321); cdc33-1 (El7a), derived from A364A (4; provided by L. Hartwell); cdc35-1 (BR214-4a; a adel ural his7 arg4 trpl); and cdc36-12 (ST21; a met2 cyh2) (provided by L. Hartwell).Media and cell growth.

The methods of centrifugal elutriation, two-dimensional gel electrophoresis, and dual isotopic labeling were applied to the study and identification of a number of purified yeast proteins. The location of polypeptide spots corresponding to specific proteins was determined on two-dimensional gels. A dual-label method was used to determine the rates of synthesis through the cell cycle of the identified proteins as well as to confirm the results of previous studies from our laboratory on unidentified proteins. The identified proteins, and the more generally defined phosphorylated, heat shock, and heat stroke proteins were found to follow the general pattern of exponential increase in rate of synthesis through the cell cycle. In addition, colorimetric enzyme activity assays were used to examine the catabolic enzyme ca-glucosidase (EC 3.2.1.20). Both the activity and synthesis of a-glucosidase were found to be nonperiodic with respect to the cell cycle. These data contrast with earlier reports of periodicity, which employed induction and selection synchrony to study enzyme expression through the yeast cell cycle.The cell cycles of eucaryotic organisms contain a number of distinct events which take place only once in each cell cycle and are restricted to a defined period. In the budding yeast Saccharomyces cerevisiae, such distinct, one-time events include: spindle pole body duplication and separation, bud emergence, DNA synthesis, nuclear migration, nuclear division, cytokinesis, and cell separation (23). In addition to distinct events, cells undergo an exponential increase in size through the cell cycle (23). In light of the temporal sequence of events through the cell cycle, one might expect to see temporal expression of specific protein products responsible for (or dependent upon) such events.Workers in this laboratory have approached the problem by applying the technique of centrifugal elutriation to the separation of asynchronous, exponentially growing populations of yeast cells into cell cycle specific fractions (14). During elutriation, cells in the separation chamber are acted upon by a centrifugal force which tends to sediment them. This force is opposed by a flow of liquid, and with increasing flow rate, cells of increasing size are washed from the chamber and collected into specific fractions. t Current address:

This paper reports the discovery and initial characterization of two small plasmids, pCf1 and pCf2, in the marine diatom Cylindrotheca fusiformis. Extracted diatom DNA separates into two bands in CsCl-Hoechst 33258 dye gradients. Upon agarose gel electrophoresis of a sample of the upper band of the gradient we observed, in addition to high molecular weight (genomic) chloroplast and mitochondrial DNA, pairs of lower molecular weight bands. These bands contained two species of circular plasmid DNA molecules, as shown by electron microscopy. The nucleotide composition of the plasmids, and chloroplast and mitochondrial DNAs is similar, as indicated by their co-banding in the gradients. They were cloned, and their restriction maps determined, showing that pCf1 is 4.27 and pCf2 4.08 kb in size. By hybridization analysis, we showed that pCf1 and pCf2 share regions of similarity, but not identity. Neither plasmid hybridizes with mitochondrial DNA. Both plasmids hybridize with chloroplast DNA, and pCf2 also hybridizes with nuclear DNA.

Platinum-based chemotherapy, such as cisplatin, is the primary treatment for ovarian cancer. However, drug resistance has become a major impediment to the successful treatment of ovarian cancer. To date, the molecular mechanisms of resistance to platinum-based chemotherapy remain unclear. In this study, we applied an LC/MS-based protein quantification method to examine the global protein expression of two pairs of ovarian cancer cell lines, A2780/A2780-CP (cisplatin-sensitive/cisplatin-resistant) and 2008/2008-C13*5.25 (cisplatin-sensitive/cisplatinresistant). We identified and quantified over 2000 proteins from these cell lines and 760 proteins showed significant expression changes with a false discovery rate of less than 5% between paired groups. Based on the results we obtained, we suggest several potential pathways that may be involved in cisplatin resistance in human ovarian cancer. This study provides not only a new proteomic platform for large-scale quantitative protein analysis, but also important information for discovery of potential biomarkers of cisplatin resistance in ovarian cancer. Furthermore, these results may be clinically relevant for diagnostics, prognostics, and therapeutic improvement for ovarian cancer treatment.

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We have discovered plasmids in 5 of 18 diatom species surveyed. In several species, more than one type of plasmid is present. Several of the plasmids show similarity by hybridization to previously characterized plasmids in Cylindrothecafusiformis (J. D. Jacobs et al., unpublished data). Additionally, there is similarity between the plasmids found in C. fusiformis and chloroplast DNA in three diatom species. These results add to the evidence that the plasmids have features of mobile genetic elements.We have recently discovered two small circular DNA plasmids, pCfl and pCf2, in the marine diatom Cylindrotheca fusiformis (9). These plasmids are 4.27 kbp (pCfl) and 4.08 kbp (pCf2) in size. Sequence analysis (8) shows that the plasmids share a large region of significant similarity. They hybridize to each other under low-stringency conditions but not under high-stringency conditions. Under high-stringency conditions, both plasmids hybridize to high-molecularweight (genomic) chloroplast DNA, and pCf2 also hybridizes to nuclear DNA. When coupled with the sequence information, this suggests that substantial portions of the plasmids are present in genomic DNA. The evidence to date is consistent with the hypothesis that the plasmids are mobile genetic elements (8). These intriguing results prompted us to see whether plasmids commonly occur in other diatom species and to determine whether they share similar features.

A study has been made of the effects of a casamino acids shift-up on a prototrophic strain of yeast growing under conditions of ammonium repression. The shift-up produced an increase in growth rate some 120 min after the addition of amino acids to the medium. This growth rate increase was slightly preceded by an increase in the rate of accumulation of DNA. In contrast, the rate of accumulation of protein increased immediately and that of RNA 15-20 min after the shift. RNA was initially accumulated at a rate greater than that required to sustain the new steady state. This was shown to be due to an increase in the rate of synthesis of the rRNA species derived from the 35S precursor. The rate of synthesis of 5S rRNA and of tRNA increased much later and to a lesser extent than that of the 35S derived species. The implications of these results for general theories of regulation of RNA synthesis are discussed.