CDC37 is required for p60v-src activity in yeast.

Mutations in genes encoding the molecular chaperones Hsp90 and Ydj1p suppress the toxicity of the protein tyrosine kinase p60v-src in yeast by reducing its levels or its kinase activity. We describe isolation and characterization of novel p60v-src-resistant, temperature-sensitive cdc37 mutants, cdc37-34 and cdc37-17, which produce less p60v-src than the parental wild-type strain at 23 degrees C. However, p60v-src levels are not low enough to account for the resistance of these strains. Asynchronously growing cdc37-34 and cdc37-17 mutants arrest in G1 and G2/M when shifted from permissive temperatures (23 degrees C) to the restrictive temperature (37 degrees C), but hydroxyurea-synchronized cdc37-34 and cdc37-17 mutants arrest in G2/M when released from the hydroxyurea block and shifted from 23 to 37 degrees C. The previously described temperature-sensitive cdc37-1 mutant is p60v-src-sensitive and produces wild-type amounts of p60v-src at permissive temperatures but becomes p60v-src-resistant at its restrictive temperature, 38 degrees C. In all three cdc37 mutants, inactivation of Cdc37p by incubation at 38 degrees C reduces p60v-src-dependent tyrosine phosphorylation of yeast proteins to low or undetectable levels. Also, p60v-src levels are enriched in urea-solubilized extracts and depleted in detergent-solubilized extracts of all three cdc37 mutants prepared from cells incubated at the restrictive temperature. These results suggest that Cdc37p is required for maintenance of p60v-src in a soluble, biologically active form.     

Mutants of Saccharomyces cerevisiae cell division cycle defective in cytokinesis. Biosynthesis of the cell wall and morphology

The four temperature-sensitive mutants of Saccharomyces cerevisiae in the cell division cycle defective in cytokinesis (cdc, 3, 10, 11 and 12), have been analyzed with respect to the biosynthesis of the cell wall polymers. After 3 hours of incubation at the non-permissive temperature (37°C) these strains stop growing. The synthesis of glucan, mannan and chitin (wall polymers) level off in a similar time, but glucan, mannan and chitin synthases remained active for at least 4 hours.If the mutants are analyzed by transmission and scanning electron microscopy different pictures emerge. Two of the mutants cdc 10 and cdc 12, after 3 hours of incubation at 37°C present apparently normal cytoplasms and cell wall surfaces with multiple elongated buds. The other two mutants, cdc 3 and cdc 11, present a completely disarranged cytoplasmic content and damage at the level of the plasma membrane is evident.These and other observations, suggest that between the execution points of cdc 3 (0.27) and cdc 10 (0.58), essential processes in the assembly of cell membrane occur.This work was supported in part by a grant from la Comisión de Investigación Científica y Técnica of the Spanish Ministerio de Educación y Ciencia (Project no. 4593-1980).     

Cell size modulation by CDC25 and RAS2 genes in Saccharomyces cerevisiae.

A detailed kinetic analysis of the cell cycle of cdc25-1, RAS2Val-19, or cdc25-1/RAS2Val-19 mutants during exponential growth is presented. At the permissive temperature (24 degrees C), cdc25-1 cells show a longer G1/unbudded phase of the cell cycle and have a smaller critical cell size required for budding without changing the growth rate in comparison to an isogenic wild type. The RAS2Val-19 mutation efficiently suppresses the ts growth defect of the cdc25-1 mutant at 36 degrees C and the increase of G1 phase at 24 degrees C. Moreover, it causes a marked increase of the critical cell mass required to enter into a new cell division cycle compared with that of the wild type. Since the critical cell mass is physiologically modulated by nutritional conditions, we have also studied the behavior of these mutants in different media. The increase in cell size caused by the RAS2Val-19 mutation is evident in all tested growth conditions, while the effect of cdc25-1 is apparently more pronounced in rich culture media. CDC25 and RAS2 gene products have been showed to control cell growth by regulating the cyclic AMP metabolic pathway. Experimental evidence reported herein suggests that the modulation of the critical cell size by CDC25 and RAS2 may involve adenylate cyclase.     

Roles of the CDC24 gene product in cellular morphogenesis during the Saccharomyces cerevisiae cell cycle

Temperature-sensitive yeast mutants defective in gene CDC24 continued to grow (i.e., increase in cell mass and cell volume) at restrictive temperature (36 degrees C) but were unable to form buds. Staining with the fluorescent dye Calcofluor showed that the mutants were also unable to form normal bud scars (the discrete chitin rings formed in the cell wall at budding sites) at 36 degrees C; instead, large amounts of chitin were deposited randomly over the surfaces of the growing unbudded cells. Labeling of cell-wall mannan with fluorescein isothiocyanate-conjugated concanavalin A suggested that mannan incorporation was also delocalized in mutant cells grown at 36 degrees C. Although the mutants have well-defined execution points just before bud emergence, inactivation of the CDC24 gene product in budded cells led both to selective growth of mother cells rather than of buds and to delocalized chitin deposition, indicating that the CDC24 gene product functions in the normal localization of growth in budded as well as in unbudded cells. Growth of the mutant strains at temperatures less than 36 degrees C revealed allele-specific differences in behavior. Two strains produced buds of abnormal shape during growth at 33 degrees C. Moreover, these same strains displayed abnormal localization of budding sites when growth at 24 degrees C (the normal permissive temperature for the mutants); in each case, the abnormal pattern of budding sites segregated with the temperature sensitivity in crosses. Thus, the CDC24 gene product seems to be involved in selection of the budding site, formation of the chitin ring at that site, the subsequent localization of new cell wall growth to the budding site and the growing bud, and the balance between tip growth and uniform growth of the bud that leads to the normal cell shape.     

Formation of septum-like structures at locations remote from the budding sites in cytokinesis-defective mutants of Saccharomyces cerevisiae.

Cell wall structures that partition membrane-bound portions of cytoplasm were formed at sites along the peripheral wall when a cytokinesis-defective cell division cycle mutant (cdc3) of Saccharomyces cerevisiae was grown at a restrictive temperature. The appearance of these structures, as observed in electron micrographs, was similar to that of normal septa. Aberrant septa were also detected in cytokinesis mutants harboring mutations cdc10, cdc11, and cdc12, after growth at 37 degrees C. Formation of the abnormal septa was abolished by the introduction, in a cdc3-containing strain, of additional cell cycle mutations that precluded events leading to cytokinesis and cell division. These results showed that septum formation can occur in the absence of cytokinesis. Formation of the abnormal structures was controlled by the same sequences of cell cycle events as formation of normal septa but was not subject to the spatial controls that ensure association of the septum with the budding site.     

cdc9 ligase-defective mutants of Saccharomyces cerevisiae exhibit lowered resistance to lethal effects of bleomycin

Conditional ligase-deficient mutants of Saccharomyces cerevisiae were more sensitive than their parental (CDC9) strain to dose-dependent killing by bleomycin, even when mutant cells were pregrown and exposed to the antibiotic at permissive temperatures. Pretreatment incubation at the restrictive temperature (37 degrees C) under growing or nongrowing conditions enhanced bleomycin killing of both cdc9-1 and cdc9-9 mutants. This sensitization could be relieved by incubation at the permissive temperature before treatment.     

Regulation of mating in the cell cycle of Saccharomyces cerevisiae

The capacity of haploid a yeast cells to mate (fuse with a haploid strain of alpha mating type followed by nuclear fusion to produce a diploid cell) was assessed for a variety of temperature-sensitive cell division cycle (cdc) mutants at the permissive and restrictive temperatures. Asynchronous populations of some mutants do not mate at the restrictive temperature, and these mutants define genes (cdc 1, 4, 24, and 33) that are essential both for the cell cycle and for mating. For most cdc mutants, asynchronous populations mate well at the restrictive temperature while populations synchronized at the cdc block do not. Populations of a mutant carrying the cdc 28 mutation mate well at the restrictive temperature after synchronization at the cdc 28 step. These results suggest that mating can occur from the cdc 28 step, the same step at which mating factors arrest cell cycle progress. The cell cycle interval in which mating can occur may or may not extend to the immediately succeeding and diverging steps (cdc 4 and cdc 24). High frequency mating does not occur in the interval of the cell cycle extending from the step before the initiation of DNA synthesis (cdc 7) through DNA synthesis (cdc 2, 8, and 21), medial nuclear division (cdc 13), and late nuclear division (cdc 14 and 15).     

The cdc30 mutation in Saccharomyces cerevisiae results in a temperature-sensitive isoenzyme of phosphoglucose isomerase

The cdc30 mutation in the yeast Saccharomyces cerevisiae causes cell cycle arrest late in nuclear division when cells are shifted from the permissive temperature of 25 degrees C to the restrictive temperature of 36.5 degrees C. Cell cycle arrest at 36.5 degrees C is dependent upon the carbon source used: a shift-up in glucose containing media results in cell cycle blockade, whereas a shift-up in ethanol, fructose, glycerol, glycerol plus ethanol, or mannose does not. Metabolite analyses showed accumulation of glucose 6-phosphate in a cdc30-bearing strain after a temperature shift-up in glucose-containing medium. Thermal denaturation studies and kinetic measurements indicate the existence of two isoenzymes of phosphoglucose isomerase (EC 5.3.1.9); one of which is apparently altered in the temperature-sensitive cell cycle mutant. We propose that the gene products of both the CDC30 and PG11 genes are required for cell cycle progression in glucose media and that the PGI1 gene product has a regulatory function over the CDC30 gene product.     

NMR analysis of a cell division cycle mutant of Saccharomyces cerevisiae

cdc 19.1 is a temperature-sensitive lesion in the genome of Saccharomyces cerevisiae. The phenotype of this mutant is a cell cycle specific arrest in G1, which is expressed at 37 degrees C. In the present study, 31P- and 13C-NMR spectroscopy were used to analyze the metabolism of the mutant at the permissive and restrictive temperatures. Our results confirm previous findings which have indicated that cdc 19.1 contains temperature-sensitive pyruvate kinase activity. In contrast to previous findings, however, the present investigation demonstrates that restriction of pyruvate kinase activity in vivo takes as long as 24 h to be fully expressed. In addition, analysis by NMR has allowed us to assess the metabolic consequences of pyruvate kinase restriction which may contribute to the arrest of cell growth in the early G1 phase of the cell division cycle.     

Cdc48 and cofactors Npl4-Ufd1 are important for G1 progression during heat stress by maintaining cell wall integrity in Saccharomyces cerevisiae

The ubiquitin-selective chaperone Cdc48, a member of the AAA (ATPase Associated with various cellular Activities) ATPase superfamily, is involved in many processes, including endoplasmic reticulum-associated degradation (ERAD), ubiquitin- and proteasome-mediated protein degradation, and mitosis. Although Cdc48 was originally isolated as a cell cycle mutant in the budding yeast Saccharomyces cerevisiae, its cell cycle functions have not been well appreciated. We found that temperature-sensitive cdc48-3 mutant is largely arrested at mitosis at 37°C, whereas the mutant is also delayed in G1 progression at 38.5°C. Reporter assays show that the promoter activity of G1 cyclin CLN1, but not CLN2, is reduced in cdc48-3 at 38.5°C. The cofactor npl4-1 and ufd1-2 mutants also exhibit G1 delay and reduced CLN1 promoter activity at 38.5°C, suggesting that Npl4-Ufd1 complex mediates the function of Cdc48 at G1. The G1 delay of cdc48-3 at 38.5°C is a consequence of cell wall defect that over-activates Mpk1, a MAPK family member important for cell wall integrity in response to stress conditions including heat shock. cdc48-3 is hypersensitive to cell wall perturbing agents and is synthetic-sick with mutations in the cell wall integrity signaling pathway. Our results suggest that the cell wall defect in cdc48-3 is exacerbated by heat shock, which sustains Mpk1 activity to block G1 progression. Thus, Cdc48-Npl4-Ufd1 is important for the maintenance of cell wall integrity in order for normal cell growth and division.     

p56(chk1) protein kinase is required for the DNA replication checkpoint at 37 degrees C in fission yeast.

Fission yeast p56(chk1) kinase is known to be involved in the DNA damage checkpoint but not to be required for cell cycle arrest following exposure to the DNA replication inhibitor hydroxyurea (HU). For this reason, p56(chk1) is considered not to be necessary for the DNA replication checkpoint which acts through the inhibitory phosphorylation of p34(cdc2) kinase activity. In a search for Schizosaccharomyces pombe mutants that abolish the S phase cell cycle arrest of a thermosensitive DNA polymerase delta strain at 37 degrees C, we isolated two chk1 alleles. These alleles are proficient for the DNA damage checkpoint, but induce mitotic catastrophe in several S phase thermosensitive mutants. We show that the mitotic catastrophe correlates with a decreased level of tyrosine phosphorylation of p34(cdc2). In addition, we found that the deletion of chk1 and the chk1 alleles abolish the cell cycle arrest and induce mitotic catastrophe in cells exposed to HU, if the cells are grown at 37 degrees C. These findings suggest that chk1 is important for the maintenance of the DNA replication checkpoint in S phase thermosensitive mutants and that the p56(chk1) kinase must possess a novel function that prevents premature activation of p34(cdc2) kinase under conditions of impaired DNA replication at 37 degrees C.     

Exo1 and Rad24 differentially regulate generation of ssDNA at telomeres of Saccharomyces cerevisiae cdc13-1 mutants

Cell cycle arrest in response to DNA damage depends upon coordinated interactions between DNA repair and checkpoint pathways. Here we examine the role of DNA repair and checkpoint genes in responding to unprotected telomeres in budding yeast cdc13-1 mutants. We show that Exo1 is unique among the repair genes tested because like Rad9 and Rad24 checkpoint proteins, Exo1 inhibits the growth of cdc13-1 mutants at the semipermissive temperatures. In contrast Mre11, Rad50, Xrs2, and Rad27 contribute to the vitality of cdc13-1 strains grown at permissive temperatures, while Din7, Msh2, Nuc1, Rad2, Rad52, and Yen1 show no effect. Exo1 is not required for cell cycle arrest of cdc13-1 mutants at 36 degrees but is required to maintain arrest. Exo1 affects but is not essential for the production of ssDNA in subtelomeric Y' repeats of cdc13-1 mutants. However, Exo1 is critical for generating ssDNA in subtelomeric X repeats and internal single-copy sequences. Surprisingly, and in contrast to Rad24, Exo1 is not essential to generate ssDNA in X or single-copy sequences in cdc13-1 rad9Delta mutants. We conclude that Rad24 and Exo1 regulate nucleases with different properties at uncapped telomeres and propose a model to explain our findings.     

Biochemical and genetic studies on the function of, and relationship between, the PGI1- and CDC30-encoded phosphoglucose isomerases in Saccharomyces cerevisiae

Isoelectric focusing was used to compare the complement of phosphoglucose isomerase isoenzymes in a wild-type strain of Saccharomyces cerevisiae and in a strain with a deletion in the PGI1 structural gene. Deletion of the PGI1 gene did not result in the absence of the high-Km isoenzyme I but the low-Km isoenzyme II was absent. Hence, the isoenzymes must be the products of two genes. If PGI1 were the sole structural gene its deletion would result in the disappearance of both isoenzymes. After a temperature shift-up a cdc30-bearing strain had cell cycle arrested and contained only 8% of the polysaccharide in the wild-type. Phosphoglucose isomerase is required for the synthesis of fructose 6-phosphate (F6-P), a precursor of the cell wall components chitin and mannoprotein ('mannan'), which are a polysaccharide and contain polysaccharide, respectively. Since the cdc30 mutation confers a temperature-sensitive phosphoglucose isomerase, the likely explanation for cell cycle arrest caused by this mutation is that the defective phosphoglucose isomerase results in a reduction of F6-P and hence an inability to synthesize the mannan and chitin needed for cytokinesis and cell separation. Revertants of a pgi1-1 bearing strain were selected for their ability to grow on glucose at 25 degrees C and this yielded a number of different phenotypes. Amongst the isolates was a strain which had undergone an intragenic reversion at the pgi1 locus, designated pgi1-1,100. This mutation permits growth and cell division at 25 degrees C but results in cell cycle arrest at 36 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rho1p mutations specific for regulation of beta(1-->3)glucan synthesis and the order of assembly of the yeast cell wall

In the yeast Saccharomyces cerevisiae, the GTP-binding protein Rho1 is required for beta(1-->3)glucan synthase activity, for activation of protein kinase C and the cell integrity pathway and for progression in G1, cell polarization and exocytosis. A genetic screen for cells that become permeabilized at non-permissive temperature was used to isolate in vitro-generated mutants of Rho1p. After undergoing a battery of tests, several of them appeared to be specifically defective in the beta(1-->3) glucan synthesis function of Rho1p. At the non-permissive temperature (37 degrees C), the mutants developed defects in the cell wall, especially at the tip of new buds. In the yeast cell wall, beta(1-->6)glucan is linked to both beta(1-->3)glucan and mannoprotein, as well as occasionally to chitin. We have used the rho1 mutants to study the order of assembly of the cell wall components. The incorporation of [(14)C]-glucose into beta(1-->3)glucan at 37 degrees C was decreased or abolished in the mutants. Concomitantly, a partial defect in the incorporation of label into cell wall mannoproteins and beta(1-->6)glucan was observed. In contrast, YW3458, an inhibitor of glycosylphosphatidylinositol anchor formation, prevented mannoprotein incorporation, whereas the beta(1-->3)-beta(1-->6)glucan complex was synthesized at almost normal levels. As beta(1-->3)glucan can be synthesized in vitro or in vivo independently, we conclude that the order of addition in vivo is beta(1-->3)glucan, beta(1-->6)glucan, mannoprotein. Previous observations indicate that chitin is the last component to be incorporated into the complex.     

The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle

The morphology of three Saccharomyces cerevisiae strains, all lacking chitin synthase 1 (Chs1) and two of them deficient in either Chs3 (calR1 mutation) or Chs2 was observed by light and electron microscopy. Cells deficient in Chs2 showed clumpy growth and aberrant shape and size. Their septa were very thick; the primary septum was absent. Staining with WGA-gold complexes revealed a diffuse distribution of chitin in the septum, whereas chitin was normally located at the neck between mother cell and bud and in the wall of mother cells. Strains deficient in Chs3 exhibited minor abnormalities in budding pattern and shape. Their septa were thin and trilaminar. Staining for chitin revealed a thin line of the polysaccharide along the primary septum; no chitin was present elsewhere in the wall. Therefore, Chs2 is specific for primary septum formation, whereas Chs3 is responsible for chitin in the ring at bud emergence and in the cell wall. Chs3 is also required for chitin synthesized in the presence of alpha-pheromone or deposited in the cell wall of cdc mutants at nonpermissive temperature, and for chitosan in spore walls. Genetic evidence indicated that a mutant lacking all three chitin synthases was inviable; this was confirmed by constructing a triple mutant rescued by a plasmid carrying a CHS2 gene under control of a GAL1 promoter. Transfer of the mutant from galactose to glucose resulted in cell division arrest followed by cell death. We conclude that some chitin synthesis is essential for viability of yeast cells.     

A novel yeast mutant that is defective in regulation of the Anaphase-Promoting Complex by the spindle damage checkpoint

The accurate segregation of sister chromatids at the metaphase to anaphase transition in Saccharomyces cerevisiae is regulated by the activity of the anaphase-promoting complex or cyclosome (APC/C). In the event of spindle damage or monopolar spindle attachment, the spindle checkpoint is activated and inhibits APC/C activity towards the anaphase inhibitor Pds1p, resulting in a cell cycle arrest at metaphase. We have identified a novel allele of a gene for an APC/C subunit, cdc16-183 , in S. cerevisiae. cdc16-183 mutants arrest at metaphase at 37°C, and are supersensitive to the spindle-damaging agent nocodazole, which activates the spindle checkpoint, at lower temperatures. This supersensitivity to nocodazole cannot be explained by impairment of the spindle checkpoint pathway, as cells respond normally to spindle damage with a stable metaphase arrest and high levels of Pds1p. Despite showing metaphase arrest at G2/M at 37°C, cdc16-183 mutants are able to perform tested G1 functions normally at this temperature. This is the first demonstration that a mutation in a core APC/C subunit can result in a MAD2-dependent arrest at the restrictive temperature. Our results suggest that the cdc16-183 mutant may have a novel APC/C defect(s) that mimics or activates the spindle checkpoint pathway.Communicated by C. P. Hollenberg     

Crh1p and Crh2p are required for the cross-linking of chitin to beta(1-6)glucan in the Saccharomyces cerevisiae cell wall

In budding yeast, chitin is found in three locations: at the primary septum, largely in free form, at the mother-bud neck, partially linked to beta(1-3)glucan, and in the lateral wall, attached in part to beta(1-6)glucan. By using a recently developed strategy for the study of cell wall cross-links, we have found that chitin linked to beta(1-6)glucan is diminished in mutants of the CRH1 or the CRH2/UTR2 gene and completely absent in a double mutant. This indicates that Crh1p and Crh2p, homologues of glycosyltransferases, ferry chitin chains from chitin synthase III to beta(1-6)glucan. Deletion of CRH1 and/or CRH2 aggravated the defects of fks1Delta and gas1Delta mutants, which are impaired in cell wall synthesis. A temperature shift from 30 degrees C to 38 degrees C increased the proportion of chitin attached to beta(1-6)glucan. The expression of CRH1, but not that of CRH2, was also higher at 38 degrees C in a manner dependent on the cell integrity pathway. Furthermore, the localization of both Crh1p and Crh2p at the cell cortex, the area where the chitin-beta(1-6)glucan complex is found, was greatly enhanced at 38 degrees C. Crh1p and Crh2p are the first proteins directly implicated in the formation of cross-links between cell wall components in fungi.     

Ultrastructure and cell wall composition in cell division cycle mutants ofSchizosaccharomyces pombe deficient in septum formation

A number of temperature-sensitive cdc^- mutants ofSchizosaccharomyces pombe that are affected in septum formation were analyzed with respect to their ultrastructure and the composition of their cell wall polymers. One mutant strain, cdc 16–116, has a cell wall composition similar to the wild type (strain 972 h^-). However two other mutants, cdc 4 and cdc 7, show a higher galactomannan content and a lower -glucan content. In all the mutants tested, total glucose incorporation, protein, RNA and DNA synthesis increased similarly to wild type over 3 1/2 h. After 2–3 h of incubation at the non permissive temperature-35°C-, cell numbers remained constant although, increases in optical densities at 600 nm were observed. According to scanning electron microscopy, the mutants had aberrant shapes after 5h of incubation at 35°C. Transmission electron microscopy showed that cdc 3 is unable to complete septum formation. cdc 4 showed the most varied morphological shapes and aberrant depositions of cell wall material. cdc 8 exhibited a deranged plasma membrane and cell wall regions near of cell poles; an abnormal septum and several nuclei. cdc 7 showed elongated cells with several nuclei and with an apparently normal cell wall completely lacking in septum and septal material. cdc 16 showed more than one septum per cell.     

Rise in intracellular pH is concurrent with 'start' progression of Saccharomyces cerevisiae

Intracellular pH (pHi) was determined during arrest and recovery of temperature sensitive-cell division cycle mutants of Saccharomyces cerevisiae. In all mutants, pHi decreased during arrest; but when the mutants were released from arrest a rapid increase in pHi ensued in only cdc28- and cdc37-arrested cells. Both of these mutations cause arrest at 'start', the sole regulatory point in the S. cerevisiae cell cycle. In cells with cdc4 or cdc7 mutations, which arrest past start, pHi remained constant and exhibited a decrease, respectively, upon recovery of growth. The activity of plasma membrane ATPase decreased during the first 30 min of recovery of cdc28-arrested cells, concomitant with the rise in pHi. During the same period, there was no significant change in activity in cdc4-bearing cells, whereas an increase was observed for cdc7-bearing cells. Increase in pHi may be used as a specific signal by S. cerevisiae for start traversal and commitment to a new cycle.     

Protein synthesis requirements for nuclear division, cytokinesis, and cell separation in Saccharomyces cerevisiae.

Protein synthesis inhibitors have often been used to identify regulatory steps in cell division. We used cell division cycle mutants of the yeast Saccharomyces cerevisiae and two chemical inhibitors of translation to investigate the requirements for protein synthesis for completing landmark events after the G1 phase of the cell cycle. We show, using cdc2, cdc6, cdc7, cdc8, cdc17 (38 degrees C), and cdc21 (also named tmp1) mutants, that cells arrested in S phase complete DNA synthesis but cannot complete nuclear division if protein synthesis is inhibited. In contrast, we show, using cdc16, cdc17 (36 degrees C), cdc20, cdc23, and nocodazole treatment, that cells that arrest in the G2 stage complete nuclear division in the absence of protein synthesis. Protein synthesis is required late in the cell cycle to complete cytokinesis and cell separation. These studies show that there are requirements for protein synthesis in the cell cycle, after G1, that are restricted to two discrete intervals.