Killer double-stranded ribonucleic acid synthesis in cell division cycle mutants of Saccharomyces cerevisiae.

The synthesis of killer double-stranded ribonucleic acid (dsRNA) in Saccharomyces cerevisiae was examined in seven different cell division cycle mutants (cdc) that are defective in nuclear deoxyribonucleic acid replication and contain the "killer character." In cdc28, cdc4, and cdc7, which are defective in the initiation of nuclear deoxyribonucleic acid synthesis, and in cdc23 or in cdc14, defective in medial or late nuclear division, an overproduction of dsRNA at the restrictive temperature was observed. In contrast to the above mutants, the synthesis of killer dsRNA is not enhanced at the restrictive temperature in either cdc8 or cdc21, which are defective in deoxyribonucleic acid chain elongation. Examination of killer sensitive strains (cdc7 K- and cdc4 K-) has shown that the complete killer dsRNA genome is essential for the overproduction of dsRNA at the restrictive temperature.     

The Role of S. CEREVISIAE Cell Division Cycle Genes in Nuclear Fusion

Forty temperature-sensitive cell division cycle (cdc) mutants of Saccharomyces cerevisiae were examined for their ability to complete nuclear fusion during conjugation in crosses to a CDC parent strain at the restrictive temperature. Most of the cdc mutant alleles behaved as the CDC parent strain from which they were derived, in that zygotes produced predominantly diploid progeny with only a small fraction of zygotes giving rise to haploid progeny (cytoductants) that signalled a failure in nuclear fusion. However, cdc4 mutants exhibited a strong nuclear fusion (karyogamy) defect in crosses to a CDC parent and cdc28, cdc34 and cdc37 mutants exhibited a weak karyogamy defect. For all four mutants, the karyogamy defect and the cell cycle defect cosegregated, suggesting that both defects resulted from a single lesion for each of these cdc mutants. Therefore, the cdc 4, 28, 34 and 37 gene products are required in both cell division and karyogamy.     

Mating-defective ste mutations are suppressed by cell division cycle start mutations in Saccharomyces cerevisiae.

Temperature-sensitive mutants which arrest in the G1 phase of the cell cycle have been described for the yeast Saccharomyces cerevisiae. One class of these mutants (carrying cdc28, cdc36, cdc37, or cdc39) forms a shmoo morphology at restrictive temperature, characteristic of mating pheromone-arrested wild-type cells. Therefore, one hypothesis to explain the control of cell division by mating factors states that mating pheromones arrest wild-type cells by inactivating one or more of these CDC gene products. A class of mutants (carrying ste4, ste5, ste7, ste11, or ste12) which is insensitive to mating pheromone and sterile has also been described. One possible function of the STE gene products is the inactivation of the CDC gene products in the presence of a mating pheromone. A model incorporating these two hypotheses predicts that such STE gene products will not be required for mating in strains carrying an appropriate cdc lesion. This prediction was tested by assaying the mating abilities of double mutants for all of the pairwise combinations of cdc and ste mutations. Lesions in either cdc36 or cdc39 suppressed the mating defect due to ste4 and ste5. Allele specificity was observed in the suppression of both ste4 and ste5. The results indicate that the CDC36, CDC39, STE4, and STE5 gene products interact functionally or physically or both in the regulation of cell division mediated by the presence or absence of mating pheromones. The cdc36 and cdc39 mutations did not suppress ste7, ste11, or ste12. Lesions in cdc28 or cdc37 did not suppress any of the ste mutations. Other models of CDC and STE gene action which predicted that some of the cdc and ste mutations would be alleles of the same locus were tested. None of the cdc mutations was allelic to the ste mutations and, therefore, these models were eliminated.     

Test for temporal or spatial restrictions in gene product function during the cell division cycle.

The ability of a functional gene to complement a nonfunctional gene may depend upon the intracellular relationship of the two genes. If so, the function of the gene product in question must be limited in time or in space. CDC (cell division cycle) gene products of Saccharomyces cerevisiae control discrete steps in cell division; therefore, they constitute reasonable candidates for genes that function with temporal or spatial restrictions. In an attempt to reveal such restrictions, we compared the ability of a CDC gene to complement a temperature-sensitive cdc gene in diploids where the genes are located within the same nucleus to complementation in heterokaryons where the genes are located in different nuclei. In CDC X cdc matings, complementation was monitored in rare heterokaryons by assaying the production of cdc haploid progeny (cytoductants) at the restrictive temperature. The production of cdc cytoductants indicates that the cdc nucleus was able to complete cell division at the restrictive temperature and implies that the CDC gene product was provided by the other nucleus or by cytoplasm in the heterokaryon. Cytoductants from cdc28 or cdc37 crosses were not efficiently produced, suggesting that these two genes are restricted spatially or temporally in their function. We found that of the cdc mutants tested 33 were complemented; cdc cytoductants were recovered at least as frequently as CDC cytoductants. A particularly interesting example was provided by the CDC4 gene. Mutations in CDC4 were found previously to produce a defect in both cell division and karyogamy. Surprisingly, the cell division defect of cdc4 nuclei is complemented by CDC4 nuclei in a heterokaryon, whereas the karyogamy defect is not.     

The Selection of S. CEREVISIAE Mutants Defective in the Start Event of Cell Division

Thirty-three temperature-sensitive mutations defective in the start event of the cell division cycle of Saccharomyces cerevisiae were isolated and subjected to preliminary characterization. Complementation studies assigned these mutations to four complementation groups, one of which, cdc28, has been described previously. Genetic analysis revealed that these complementation groups define single nuclear genes, unlinked to one another. One of the three newly identified genes, cdc37, has been located in the yeast linkage map on chromosome IV, two meiotic map units distal to hom2.—Each mutation produces stage-specific arrest of cell division at start, the same point where mating pheromone interrupts division. After synchronization at start by incubation at the restrictive temperature, the mutants retain the capacity to enlarge and to conjugate.     

Characterization of the energy-dependent, mating factor-activated Ca influx in Saccharomyces cerevisiae

The yeast mating pheromones, a and alpha factors, bind to specific G protein-coupled receptors in haploid cells and bring about both growth arrest in the early G1 phase of the cell cycle and differentiation into mating capable cells. This induces an increase in Ca2+ influx leading to elevated intracellular calcium concentrations, which has been shown to be essential for subsequent downstream events and the mating process itself [1]. We have characterized the alpha factor induced increase in cellular Ca2+ in wild type S. cerevisiae and in the temperature-sensitive cell division cycle mutants cdc7 and cdc28 which are growth-arrested at the G0-G1 border at the nonpermissive temperature. We observed a 2-4 fold increase in the initial velocity of Ca2+ influx in alpha factor-treated wild-type cells and in cdc7 and cdc28 cells grown at the nonpermissive temperature. Calcium influx was energy dependent, inhibited by membrane depolarization and slightly increased by hyperpolarization. Furthermore, Ca2+ influx was sensitive to both divalent and trivalent cations, but was unaffected by nifedipine and verapamil. These data demonstrate that budding yeast possesses a regulated Ca2+ transport mechanism, the activation of which is dependent upon exit out of the cell cycle and growth cessation. This transport mechanism has many similarities to that observed in mitogen-stimulated mammalian cells.     

Cell Cycle Arrest of Cdc Mutants and Specificity of the Rad9 Checkpoint

In eucaryotes a cell cycle control called a checkpoint ensures that mitosis occurs only after chromosomes are completely replicated and any damage is repaired. The function of this checkpoint in budding yeast requires the RAD9 gene. Here we examine the role of the RAD9 gene in the arrest of the 12 cell division cycle (cdc) mutants, temperature-sensitive lethal mutants that arrest in specific phases of the cell cycle at a restrictive temperature. We found that in four cdc mutants the cdc rad9 cells failed to arrest after a shift to the restrictive temperature, rather they continued cell division and died rapidly, whereas the cdc RAD cells arrested and remained viable. The cell cycle and genetic phenotypes of the 12 cdc RAD mutants indicate the function of the RAD9 checkpoint is phase-specific and signal-specific. First, the four cdc RAD mutants that required RAD9 each arrested in the late S/G(2) phase after a shift to the restrictive temperature when DNA replication was complete or nearly complete, and second, each leaves DNA lesions when the CDC gene product is limiting for cell division. Three of the four CDC genes are known to encode DNA replication enzymes. We found that the RAD17 gene is also essential for the function of the RAD9 checkpoint because it is required for phase-specific arrest of the same four cdc mutants. We also show that both X- or UV-irradiated cells require the RAD9 and RAD17 genes for delay in the G(2) phase. Together, these results indicate that the RAD9 checkpoint is apparently activated only by DNA lesions and arrests cell division only in the late S/G(2) phase.     

Meiotic Recombination and DNA Synthesis in a New Cell Cycle Mutant of SACCHAROMYCES CEREVISIAE

Vegetative cells carrying the new temperature-sensitive mutation cdc40 arrest at the restrictive temperature with a medial nuclear division phenotype. DNA replication is observed under these conditions, but most cells remain sensitive to hydroxyurea and do not complete the ongoing cell cycle if the drug is present during release from the temperature block. It is suggested that the cdc40 lesion affects an essential function in DNA synthesis. Normal meiosis is observed at the permissive temperature in cdc40 homozygotes. At the restrictive temperature, a full round of premeiotic DNA replication is observed, but neither commitment to recombination nor later meiotic events occur. Meiotic cells that are already committed to the recombination process at the permissive temperature do not complete it if transferred to the restrictive temperature before recombination is realized. These temperature shift-up experiments demonstrate that the CDC40 function is required for the completion of recombination events, as well as for the earlier stage of recombination commitment. Temperature shift-down experiments with cdc40 homozygotes suggest that meiotic segregation depends on the final events of recombination rather than on commitment to recombination.     

A dependent pathway of gene functions leading to chromosome segregation in Saccharomyces cerevisiae

Methyl-benzimidazole-2-ylcarbamate (MBC) inhibits the mitotic cell cycle of Saccharomyces cerevisiae at a stage subsequent to DNA synthesis and before the completion of nuclear division (Quinlan, R. A., C. I. Pogson, and K, Gull, 1980, J Cell Sci., 46: 341-352). The step in the cell cycle that is sensitive to MBC inhibition was ordered to reciprocal shift experiments with respect to the step catalyzed by cdc gene products. Execution of the CDC7 step is required for the initiation of DNA synthesis and for completion of the MBC-sensitive step. Results obtained with mutants (cdc2, 6, 8, 9, and 21) defective in DNA replication and with an inhibitor of DNA replication (hydroxyurea) suggest that some DNA replication required for execution of the MBC-sensitive step but that the completion of replication is not. Of particular interest were mutants (cdc5, 13, 14, 15, 16, 17, and 23) that arrest cell division after DNA replication but before nuclear division since previous experiments had not been able to resolve the pathway of events in this part of the cell cycle. Execution of the CDC17 step was found to be a prerequisite for execution of the MBC- sensitive step; the CDC13, 16 and 23 steps are executed independently of the MBC-sensitive step; execution of the MBC-sensitive step is prerequisite for execution of the MBC-sensitive step; execution of the MBC-sensitive step is prerequisite for execution of the CDC14 and 23 steps. These results considerably extend the dependent pathway of events that constitute the cell cycle of S. cerevisiae.     

Mutations in cell division cycle genes CDC36 and CDC39 activate the Saccharomyces cerevisiae mating pheromone response pathway.

Conditional mutations in the genes CDC36 and CDC39 cause arrest in the G1 phase of the Saccharomyces cerevisiae cell cycle at the restrictive temperature. We present evidence that this arrest is a consequence of a mutational activation of the mating pheromone response. cdc36 and cdc39 mutants expressed pheromone-inducible genes in the absence of pheromone and conjugated in the absence of a mating pheromone receptor. On the other hand, cells lacking the G beta subunit or overproducing the G alpha subunit of the transducing G protein that couples the receptor to the pheromone response pathway prevented constitutive activation of the pathway in cdc36 and cdc39 mutants. These epistasis relationships imply that the CDC36 and CDC39 gene products act at the level of the transducing G protein. The CDC36 and CDC39 gene products have a role in cellular processes other than the mating pheromone response. A mating-type heterozygous diploid cell, homozygous for either the cdc36 or cdc39 mutation, does not exhibit the G1 arrest phenotype but arrests asynchronously with respect to the cell cycle. A similar asynchronous arrest was observed in cdc36 and cdc39 cells where the pheromone response pathway had been inactivated by mutations in the transducing G protein. Furthermore, cdc36 and cdc39 mutants, when grown on carbon catabolite-derepressing medium, did not arrest in G1 and did not induce pheromone-specific genes at the restrictive temperature.     

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.     

Three additional genes required for deoxyribonucleic acid synthesis in Saccharomyces cerevisiae

Deoxyribonucleic acid (DNA) synthesis was examined in asynchronous and synchronous cultures of a number of cdc (cell division cycle) temperature-sensitive mutant strains. The kinetics of DNA synthesis after a shift to the restrictive temperature was compared with that obtained after inhibition of protein synthesis at the permissive temperature, a condition that specifically blocks the initiation of new rounds of DNA replication, but does not block those in progress. Mutations in three genes (cdc 4, 7, and 28) appear to block a precondition for DNA synthesis since cells carrying these lesions cannot start new rounds of DNA replication after a shift from permissive to restrictive temperature, but can finish rounds that were in progress. These three genes are classified as having roles in the "initiation" of DNA synthesis. Mutations in two genes (cdc 8 and 21) block DNA synthesis, itself, since cells harboring these lesions that had started DNA synthesis at the permissive temperature arrest synthesis abruptly upon a shift to the restrictive temperature. Mutations in 13 other cdc genes do not impair DNA synthesis in the first cell cycle at the restrictive temperature.     

Mutants of Saccharomyces cerevisiae unresponsive to cell division control by polypeptide mating hormone

Temperature-sensitive mutations that produce insensitivity to division arrest by alpha-factor, a mating pheromone, were isolated in an MATa strain of Saccharomyces cerevisiae and shown by complementation studies to difine eight genes. All of these mutations (designated ste) produce sterility at the restrictive temperature in MATa cells, and mutations in seven of the genes produce sterility in MAT alpha cells. In no case was the sterility associated with these mutations coorectible by including wild-type cells of the same mating type in the mating test nor did nay of the mutants inhibit mating of the wild-type cells; the defect appears to be intrinsic to the cell for mutations in each of the genes. Apparently, none of the mutants is defective exclusively in division arrest by alpha-factor, as the sterility of none is suppressed by a temperature-sensitive cdc 28 mutation (the latter imposes division arrest at the correct cell cycle stage for mating). The mutants were examined for features that are inducible in MATa cells by alpha-factor (agglutinin synthesis as well as division arrest) and for the characteristics that constitutively distinguish MATa from MAT alpha cells (a-factor production, alpha-factor destruction). ste2 Mutants are defective specifically in the two inducible properties, whereas ste4, 5, 7, 8, 9, 11, and 12 mutants are defective, to varying degrees, in constitutive as well as inducible aspects. Mutations in ste8 and 9 assume a polar budding pattern unlike either MATa or MAT alpha cells but characteristic of MATa/alpha cells. This study defines seven genes that function in two cell types (MATa and alpha) to control the differentiation of cell type and one gene, ste2, that functions exclusively in MATa cells to mediate responsiveness to polypeptide hormone.     

Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe

Summary Twenty seven recessive temperature sensitive mutants have been isolated in Schizosaccharomyces pombe which are unable to complete the cell division cycle at the restrictive temperature. These mutants define 14 unlinked genes which are involved in DNA synthesis, nuclear division and cell plate formation. The products from most of these genes complete their function just before the cell cycle event in which they are involved. Physiological characterisation of the mutants has shown that DNA synthesis and nuclear division form a cycle of mutually dependent events which can operate in the absence of cell plate formation. Cell plate formation itself is usually dependent upon the completion of nuclear division.     

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.     

Uncontrolled septation in a cell division cycle mutant of the fission yeast Schizosaccharomyces pombe.

A temperature-sensitive Schizosaccharomyces pombe mutant, cdc16-116, has been isolated which undergoes uncontrolled septation during its cell division cycle. The mutant accumulates two types of cells after 3 h of growth at the restrictive temperature: (i) type I cells (85% of the population), which complete nuclear division and then form up to five septa between the divided nuclei; and (ii) type II cells (15% of the population), which form an asymmetrically situated septum in the absence of any nuclear division. cdc16-116 is a monogenic recessive mutation unlinked to any previously known cdc gene of S. pombe. It is not affected in a previously reported control by which septation is dependent upon completion of nuclear division. We propose the cdc16-116 is unable to complete septum formation and proceed to cell separation and is also defective in a control which prevents the manufacture of more than one septum in each cell cycle.     

Cell size specific binding of the fluorescent dye calcofluor to budding yeast

The yeast Saccharomyces cerevisiae cell surface outside of the bud scars displayed an increasing fluorescence intensity with increasing cell size (volume), where fluorescence was due to irreversible binding of the fluorescent dye calcofluor. The increase in fluorescence intensity appeared to be due to an increase in the density of fluorescence per unit surface area of the cell. Exposure time measurements from a photomicroscope were used to quantitate fluorescence intensity on individual cells. The cell size dependent increase in fluorescence intensity was displayed by unbudded cells from stationary phase populations, and unbudded and parent cells from exponentially growing populations. Abnormally large cells generated during the arrest of cell division with alpha-factor or restrictive temperature for cdc3, 8, 13, 24, and 28 cell division cycle mutants, displayed significantly greater fluorescence intensity compared to the smaller cells generated during the arrest of division for cdc25, 33, and 35 mutant strains. Fluorescence intensity on newly emerging buds was broadly dependent on both the size of the bud, and the size of the parent cells on which the buds were growing.     

Defective DNA Synthesis in Permeabilized Yeast Mutants

THE simple eukaryote, Saccharomyces cerevisiae, is suitable for combined genetic and biochemical analysis of the cell division cycle. More than forty temperature-sensitive mutants of S. cerevisiae defective in fifteen genes that control various steps of the yeast cell cycle have been detected by screening a collection of mutants with time-lapse photomicroscopy¹. Mutations in two genes, cdc4 and cdc8, result in defective DNA synthesis at the restrictive temperature². The product of cdc8 is apparently required throughout the period of DNA synthesis, because if a strain defective in this gene is shifted to 36° C within the S period, DNA replication ceases. In contrast, the product of cdc4 is apparently required only at the initiation of DNA synthesis because when a strain carrying a defect in this gene is shifted to 36° C, DNA replication already in progress is not impaired. Cells defective in cdc4, however, fail to initiate new rounds of DNA synthesis at the restrictive temperature. Based on these observations the DNA mutants have been tentatively classified as defective in DNA replication (cdc8) and in the initiation of DNA synthesis (cdc4).     

Genetic Control of the Cell Division Cycle in Yeast: V. Genetic Analysis of cdc Mutants

One hundred and forty-eight temperature-sensitive cell division cycle (cdc) mutants of Saccharomyces cerevisiae have been isolated and characterized. Complementation studies ordered these recessive mutations into 32 groups and tetrad analysis revealed that each of these groups defines a single nuclear gene. Fourteen of these genes have been located on the yeast genetic map. Functionally related cistrons are not tightly clustered.

Mutations in different cistrons frequently produce different cellular and nuclear morphologies in the mutant cells following incubation at the restrictive temperature, but all the mutations in the same cistron produce essentially the same morphology. The products of these genes appear, therefore, each to function individually in a discrete step of the cell cycle and they define collectively a large number of different steps.

The mutants were examined by time-lapse photomicroscopy to determine the number of cell cycles completed at the restrictive temperature before arrest. For most mutants, cells early in the cell cycle at the time of the temperature shift (before the execution point) arrest in the first cell cycle while those later in the cycle (after the execution point) arrest in the second cell cycle. Execution points for allelic mutations that exhibit first or second cycle arrest are rather similar and appear to be cistron-specific. Other mutants traverse several cycles before arrest, and its suggested that the latter type of response may reveal gene products that are temperature-sensitive for synthesis, whereas the former may be temperature-sensitive for function.

The gene products that are defined by the cdc cistrons are essential for the completion of the cell cycle in haploids of a and α mating type and in a/α diploid cells. The same genes, therefore, control the cell cycle in each of these stages of the life cycle.

     

Specification of sites for polarized growth in Saccharomyces cerevisiae and the influence of external factors on site selection.

Many eucaryotic cell types exhibit polarized cell growth and polarized cell division at nonrandom sites. The sites of polarized growth were investigated in G1 arrested haploid Saccharomyces cerevisiae cells. When yeast cells are arrested during G1 either by treatment with alpha-factor or by shifting temperature-sensitive cdc28-1 cells to the restrictive temperature, the cells form a projection. Staining with Calcofluor reveals that in both cases the projection usually forms at axial sites (i.e., next to the previous bud scar); these are the same sites where bud formation is expected to occur. These results indicate that sites of polarized growth are specified before the end of G1. Sites of polarized growth can be influenced by external conditions. Cells grown to stationary phase and diluted into fresh medium preferentially select sites for polarized growth opposite the previous bud scar (i.e., distal sites). Incubation of cells in a mating mixture results in projection formation at nonaxial sites: presumably cells form projections toward their mating partner. These observations have important implications in understanding three aspects of cell polarity in yeast: 1) how yeast cell shape is influenced by growth conditions 2) how sites of polarized growth are chosen, and 3) the pathway by which polarity is affected and redirected during the mating process.