Control of the timing of cell division in fission yeast : Cell size mutants reveal a second control pathway

Strains of Schizosaccharomyces pombe carrying the wee 1 mutation divide at a reduced cell size compared with the wild-type. In this paper, we investigate the mechanism which determines the time of division and cell size at division in wee 1 strains, using three experimental approaches. The evidence suggests that the wild-type control (a cell size control over entry into nuclear division) is absent in wee 1 strains. Instead, a mechanism operates which comprises a cell size control over the initiation of S phase plus a minimum incompressible period in G2 (“timer”) from S phase to nuclear division. The elements of this second control mechanism exist in wild-type cells, though the control is not normally expressed. In particular, the G2 interval in wild-type cells is normally longer than that in wee 1 cells, but can be reduced to this minimum value by delaying S phase. Thus there are two independent controls over entry into nuclear division, one of which operates in wild-type, and the other in wee 1 cells.     

Mutants altered in the control co-ordinating cell division with cell growth in the fission yeast Schizosaccharomyces pombe

Summary The control co-ordinating cell division with cell growth has been investigated in the fission yeast Schizosaccharomyces pombe. Twenty-five mutants altered in this control have been isolated which have the same growth rate as wild type but divide at a smaller cell size. The mutants define two genes wee 1 and wee 2, both of which are involved in a control initiating mitosis when the cell attains a critical size.     

The RHC21 gene of budding yeast, a homologue of the fission yeast rad21 +gene, is essential for chromosome segregation

The Saccharomyces cerevisiae gene RHC21 is a homologue of the fission yeast rad21 ⁺gene, which affects the sensitivity of cells to γ-irradiation and is essential for cell growth in S. pombe. Disruption of the RHC21 gene showed that it is also essential in S. cerevisiae. To examine its function in cell growth further, we have isolated temperature-sensitive mutants for the RHC21 gene and characterized one of them, termed rhc21-sk16. When this mutant was incubated at 36° C, the percentage of large-budded cells was increased. Most of the large-budded cells had aberrant nuclear structures, such as unequally extended nuclear DNA with incompletely elongated spindles across the mother-daughter neck or only in a mother cell. Furthermore, a circular minichromosome is more unstable in the mutant than in the wild-type, even at 25° C. Flow cytometry showed that the bulk of DNA replication takes place normally at the restrictive temperature in the mutant. These results indicated that the RHC21 gene is required for proper segregation of the chromosomes. In addition, we found that the mutant is sensitive not only to UV radiation and γ-rays but also to the antimicrotubule agent nocodazole at 25° C. This suggests that the RHC21 gene is involved in the microtubule function. We discuss how the RHC21 gene product may be involved in chromosome segregation and microtubule function. Received: 10 March 1997 / Accepted: 1 September 1997     

cyd,a mutant of pea that alters embryo morphology is defective in cytokinesis

Embryogenesis in higher plants requires the precise regulation of cell division, orientation of cell elongation and specification of cell differentiation. The division plane is determined by the position of a new cell plate at cytokinesis. A mutant of pea has been isolated in which both the embryo pattern and surface morphology is altered. The phenotype of the mutant is manifest primarily in the cotyledons where cell plates only partially form, generating cell wall stubs and multinucleate cells. Some cotyledonary cells of the mutant proceed through nine DNA replication cycles, including nuclear division, but not cytokinesis, producing nuclei with a DNA content of ca. 1000C. The cytological phenotype of the mutant could be mimicked by the treatment of wild-type cells with caffeine. We have termed this mutant cytokinesis-defective (cyd). © 1995 Wiley-Liss, Inc.     

The RHC21 gene of budding yeast, a homologue of the fission yeast rad21 +gene, is essential for chromosome segregation

The Saccharomyces cerevisiae gene RHC21 is a homologue of the fission yeast rad21 ⁺gene, which affects the sensitivity of cells to γ-irradiation and is essential for cell growth in S. pombe. Disruption of the RHC21 gene showed that it is also essential in S. cerevisiae. To examine its function in cell growth further, we have isolated temperature-sensitive mutants for the RHC21 gene and characterized one of them, termed rhc21-sk16. When this mutant was incubated at 36°?C, the percentage of large-budded cells was increased. Most of the large-budded cells had aberrant nuclear structures, such as unequally extended nuclear DNA with incompletely elongated spindles across the mother-daughter neck or only in a mother cell. Furthermore, a circular minichromosome is more unstable in the mutant than in the wild-type, even at 25°?C. Flow cytometry showed that the bulk of DNA replication takes place normally at the restrictive temperature in the mutant. These results indicated that the RHC21 gene is required for proper segregation of the chromosomes. In addition, we found that the mutant is sensitive not only to UV radiation and γ-rays but also to the antimicrotubule agent nocodazole at 25°?C. This suggests that the RHC21 gene is involved in the microtubule function. We discuss how the RHC21 gene product may be involved in chromosome segregation and microtubule function.     

Differential effects of caffeine on DNA damage and replication cell cycle checkpoints in the fission yeast Schizosaccharomyces pombe

Caffeine potentiates the lethal effects of ultraviolet and ionising radiation on wild-type Schizosaccharomyces pombe cells. In previous studies this was attributed to the inhibition by caffeine of a novel DNA repair pathway in S. pombe that was absent in the budding yeast Saccharomyces cerevisiae. Studies with radiation-sensitive S. pombe mutants suggested that this caffeine-sensitive pathway could repair ultraviolet radiation damage in the absence of nucleotide excision repair. The alternative pathway was thought to be recombinational and to operate in the G₂ phase of the cell cycle. However, in this study we show that cells held in G₁ of the cell cycle can remove ultraviolet-induced lesions in the absence of nucleotide excision repair. We also show that recombination-defective mutants, and those now known to define the alternative repair pathway, still exhibit the caffeine effect. Our observations suggest that the basis of the caffeine effect is not due to direct inhibition of recombinational repair. The mutants originally thought to be involved in a caffeine-sensitive recombinational repair process are now known to be defective in arresting the cell cycle in S and/or G₂ following DNA damage or incomplete replication. The gene products may also have an additional role in a DNA repair or damage tolerance pathway. The effect of caffeine could, therefore, be due to interference with DNA damage checkpoints, or inhibition of the DNA damage repair/tolerance pathway. Using a combination of flow cytometric analysis, mitotic index analysis and fluorescence microscopy we show that caffeine interferes with intra-S phase and G₂ DNA damage checkpoints, overcoming cell cycle delays associated with damaged DNA. In contrast, caffeine has no effect on the DNA replication S phase checkpoint in reponse to inhibition of DNA synthesis by hydroxyurea. Received: 16 June 1998 / Accepted: 13 July 1998     

Long-Term Single Cell Analysis of S. pombe on a Microfluidic Microchemostat Array

Although Schyzosaccharomyces pombe is one of the principal model organisms for studying the cell cycle, surprisingly few methods have characterized S. pombe growth on the single cell level, and no methods exist capable of analyzing thousands of cells and tens of thousands of cell division events. We developed an automated microfluidic platform permitting S. pombe to be grown on-chip for several days under defined and changeable conditions. We developed an image processing pipeline to extract and quantitate several physiological parameters including cell length, time to division, and elongation rate without requiring synchronization of the culture. Over a period of 50 hours our platform analyzed over 100000 cell division events and reconstructed single cell lineages up to 10 generations in length. We characterized cell lengths and division times in a temperature shift experiment in which cells were initially grown at 30°C and transitioned to 25°C. Although cell length was identical at both temperatures at steady-state, we observed transient changes in cell length if the temperature shift took place during a critical phase of the cell cycle. We further show that cells born with normal length do divide over a wide range of cell lengths and that cell length appears to be controlled in the second generation, were large newly born cells have a tendency to divide more rapidly and thus at a normalized cell size. The platform is thus applicable to measure fine-details in cell cycle dynamics, should be a useful tool to decipher the molecular mechanism underlying size homeostasis, and will be generally applicable to study processes on the single cell level that require large numbers of precision measurements and single cell lineages.     

Structural diversity and dynamics of genomic replication origins in Schizosaccharomyces pombe

DNA replication origins (ORI) in Schizosaccharomyces pombe colocalize with adenine and thymine (A+T)‐rich regions, and earlier analyses have established a size from 0.5 to over 3 kb for a DNA fragment to drive replication in plasmid assays. We have asked what are the requirements for ORI function in the chromosomal context. By designing artificial ORIs, we have found that A+T‐rich fragments as short as 100 bp without homology to S. pombe DNA are able to initiate replication in the genome. On the other hand, functional dissection of endogenous ORIs has revealed that some of them span a few kilobases and include several modules that may be as short as 25–30 contiguous A+Ts capable of initiating replication from ectopic chromosome positions. The search for elements with these characteristics across the genome has uncovered an earlier unnoticed class of low‐efficiency ORIs that fire late during S phase. These results indicate that ORI specification and dynamics varies widely in S. pombe, ranging from very short elements to large regions reminiscent of replication initiation zones in mammals.     

Defining the Epigenetic Mechanism of Asymmetric Cell Division of Schizosaccharomyces japonicus Yeast

A key question in developmental biology addresses the mechanism of asymmetric cell division. Asymmetry is crucial for generating cellular diversity required for development in multicellular organisms. As one of the potential mechanisms, chromosomally borne epigenetic difference between sister cells that changes mating/cell type has been demonstrated only in the Schizosaccharomyces pombe fission yeast. For technical reasons, it is nearly impossible to determine the existence of such a mechanism operating during embryonic development of multicellular organisms. Our work addresses whether such an epigenetic mechanism causes asymmetric cell division in the recently sequenced fission yeast, S. japonicus (with 36% GC content), which is highly diverged from the well-studied S. pombe species (with 44% GC content). We find that the genomic location and DNA sequences of the mating-type loci of S. japonicus differ vastly from those of the S. pombe species. Remarkably however, similar to S. pombe, the S. japonicus cells switch cell/mating type after undergoing two consecutive cycles of asymmetric cell divisions: only one among four “granddaughter” cells switches. The DNA-strand–specific epigenetic imprint at the mating-type locus1 initiates the recombination event, which is required for cellular differentiation. Therefore the S. pombe and S. japonicus mating systems provide the first two examples in which the intrinsic chirality of double helical structure of DNA forms the primary determinant of asymmetric cell division. Our results show that this unique strand-specific imprinting/segregation epigenetic mechanism for asymmetric cell division is evolutionary conserved. Motivated by these findings, we speculate that DNA-strand–specific epigenetic mechanisms might have evolved to dictate asymmetric cell division in diploid, higher eukaryotes as well.     

N-glycans are not required for the efficient degradation of the mutant Saccharomyces cerevisiae CPY* in Schizosaccharomyces pombe

In eukaryotic cells, aberrant proteins generated in the endoplasmic reticulum (ER) are degraded by the ER-associated degradation (ERAD) pathway. Here, we report on the ERAD pathway of the fission yeast Schizosaccharomyces pombe. We constructed and expressed Saccharomyces cerevisiae wild-type CPY (ScCPY) and CPY-G255R mutant (ScCPY*) in S. pombe. While ScCPY was glycosylated and efficiently transported to the vacuoles in S. pombe, ScCPY* was retained in the ER and was not processed to the matured form in these cells. Cycloheximide chase experiments revealed that ScCPY* was rapidly degraded in S. pombe, and its degradation depended on Hrd1p and Ubc7p homologs. We also found that Mnl1p and Yos9p, proteins that are essential for ERAD in S. cerevisiae, were not required for ScCPY* degradation in S. pombe. Moreover, the null-glycosylation mutant of ScCPY, CPY*0000, was rapidly degraded by the ERAD pathway. These results suggested that N-linked oligosaccharides are not important for the recognition of luminal proteins for ERAD in S. pombe cells.     

Identification and characterization of a complex chromosomal replication origin in<Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

In the budding yeast,S. cerevisiae, two-dimensional (2D) gel electrophoresis techniques permit mapping of DNA replication origins to short stretches of DNA (±300 bp). In contrast, in mammalian cells andDrosophila, 2D gel techniques do not permit precise origin localization; the results have been interpreted to suggest that replication initiates in broad zones (several kbp or more). However, alternative techniques (replication timing, nascent strand polarity analysis, nascent strand size analysis) suggest that mammalian origins can be mapped to short DNA stretches, just likeS. cerevisiae origins. Because the fission yeast,Schizosaccharomyces pombe, resembles higher organisms in several ways to a greater extent than doesS. cerevisiae, we thought thatS. pombe replication origins might prove to resemble — and thus be helpful models for — animal cell origins. An attempt to test this possibility using 2D gel techiques resulted in identification of a replication origin near theura4 gene on chromosome III ofS. pombe. The 2D gel patterns produced by thisS. pombe origin indeed resemble the patterns produced by animal cell origins and show that theS. pombe origin cannot be precisely located. The data suggest an initiation zone of 3–5 kbp. Some aspects of the 2D gel patterns detected at theS. pombe origin cannot be explained by the rationale of initiation in broad zones, suggesting that future biochemical and genetic studies of this complex origin are likely to provide information useful in helping to understand the apparent conflict between the 2D gel mapping techniques and other mapping techniques at animal cell origins.     

Regulatory Genes Controlling Mitosis in the Fission Yeast SCHIZOSACCHAROMYCES POMBE

Fifty-two wee mutants that undergo mitosis and cell division at a reduced size compared with wild type have been genetically analyzed. The mutants define two genes, wee1 and cdc2, which control the timing of mitosis. Fifty-one of the mutants map at the wee1 locus, which is unlinked to any known cdc gene. One of the wee1 alleles has been shown to be nonsense suppressible. The 52nd wee mutant maps within cdc2. Previously, only temperature-sensitive mutants that become blocked at mitosis have been found at the cdc2 locus. The simplest interpretation of these observations is that wee1⁺ codes for a negative element or inhibitor, and cdc2⁺ codes for a positive element or activator in the mitotic control. The gene dosage of wee1⁺ plays some role in determining the timing of mitosis, but the gene dosage of cdc2⁺ has little effect. However, some aspect of the cdc2 gene product activity is important for determining when mitosis takes place. The possible roles of wee1 and cdc2 in the mitotic control are discussed, with particular reference to the part they may play in the monitoring of cell size and cell growth rate, both of which influence the timing of mitosis.     

Effect of heat on the viability of Schizosaccharomyces pombe 972h growing in synchronous cultures

The viability of synchronous cultures of the fission yeast Schizosaccharomyces pombe 972h⁻ has been examined after exposure to temperatures of 49 °C. Synchronous cultures were established by continuous flow size selection. Samples were taken at 20 min intervals over two cell cycles and heat shocked for 15 min. The cells showed different sensitivities to heat treatment during the cell cycle. The sensitive stage lasted from nuclear division to a point in early G2. The position in the cell cycle and duration of the heat sensitive stage of S. pombe are similar to those reported for the response of this organism to ultraviolet light, γ radiation, and to suicide labelling with ³²P.     

Molecular cloning and analysis of Schizosaccharomyces pombe rad9, a gene involved in DNA repair and mutagenesis

Summary The mutant allele rad9-192 renders Schizosaccharomyces pombe cells sensitive to ionizing radiation and UV light. We have isolated from a S. pombe genomic DNA library a unique recombinant plasmid that is capable of restoring wild-type levels of radioresistance to a rad9 192-containing cell population. Plasmid integration studies using the cloned DNA, coupled with mating and tetrad analyses, indicate that this isolated DNA contains the wild-type rad9 gene. We inactivated the repair function of the cloned fragment by a single insertion of the S. pombe ura4 gene. This nonfunctional fragment was used to create a viable disruption mutant, thus demonstrating that the rad9 gene does not encode an essential cellular function. In addition, the rad9-192 mutant population is as radiosensitive as the disruption mutant, indicating that rad9 gene function is severely if not totally inhibited by the molecular defect responsible for the rad9-192 phenotype. DNA sequence analysis of rad9 reveals an open reading frame of 1,278 bp, interrupted by three introns 53 bp, 57 bp, and 56 by long, respectively, and ending in the termination codon TAG. This gene is capable of encoding a protein of 426 amino acids, with a corresponding calculated molecular weight of 47,464 daltons. No significant homology was detected between the rad9 gene or its deduced protein sequence and sequences previously entered into DNA and protein sequence data banks.     

A new gene controlling the frequency of cell division per round of DNA replication in Escherichia coli

Summary A novel mutant of Escherichia coli, named cfcA1, was isolated from a temperature-sensitive dnaB42 strain, and found to have the following characteristics. Division arrest and lethality induced by inhibition of DNA replication was reduced and delayed in the cfcA1 dnaB42 strain, as compared with the parental dnaB42 strain. Two types of inhibition of division induced by the addition of nalidixic acid or hydroxyurea were suppressed by the cfcA1 mutation. Under permissive conditions for DNA replication, the colony forming ability of cfcA1 cells was significantly reduced as compared with that of cfc ⁺ cells; conversely the division rate of cfcA1 cells was higher than that of cfc ⁺ cells. The cfcA1 mutation partially restored division arrest induced in the thermosensitive ftsZ84 mutant at the restrictive temperature and suppresed the UV sensitivity of the lon mutation. The mutation was mapped at 79.2 min on the E. coli chromosome. Taking these properties into account, it is hypothesized that the cfcA gene is involved in determining the frequency of cell division per round of DNA replication by interacting with the FtsZ protein which is essential for cell division.     

Checkpoint effects and telomere amplification during DNA re-replication in fission yeast

Background  

Although much is known about molecular mechanisms that prevent re-initiation of DNA replication on newly replicated DNA during a single cell cycle, knowledge is sparse regarding the regions that are most susceptible to re-replication when those mechanisms are bypassed and regarding the extents to which checkpoint pathways modulate re-replication. We used microarrays to learn more about these issues in wild-type and checkpoint-mutant cells of the fission yeast, Schizosaccharomyces pombe.     

Genetic evidence that aphidicolin inhibits in vivo DNA synthesis in Chinese hamster ovary cells

Using a genetic approach, Chinese hamster ovary (CHO) cells sensitive (aph^S) and resistant (aph^R) to aphidicolin were grown in the presence or absence of various DNA polymerase inhibitors, and the newly synthesized DNA isolated from [³²P]dNMP-labelled, detergent-permeabilized cells, was characterized after fractionation by gel electrophoresis. The particular aph ^Rmutant CHO cell line used was one selected for resistance to aphidicolin and found to possess an altered DNA polymerase of the a-family. The synthesis of a 24 kb replication intermediate was inhibited in wild-type CHO cells grown in the presence of aphidicolin, whereas the synthesis of this replication intermediate was not inhibited by this drug in the mutant CHO cells or in the aphidicolin-resistant somatic cell hybrid progeny constructed by fusion of wild-type and mutant cell lines. Arabinofuranosylcytosine (ara-C), like aphidicolin, inhibited the synthesis of this 24 kb DNA replication intermediate in the wild-type CHO cells but not in the aph^R mutant cells. However, carbonyldiphosphonate (COMDP) inhibited the synthesis of the 24 kb replication intermediate in both wild-type and mutant cells. N²-(p-n-Butylphenyl)-2 deoxyguanisine-5-triphosphate (BuPdGTP) was found to inhibit the formation of Okazaki fragments equally well in the wild-type and mutant cell lines and thus led to inhibition of synthesis of DNA intermediates in both cases. It appears that aphidicolin and ara-C both affect a common target on the DNA polymerase, which is different from that affected by COMDP in vivo. These data also show that aphidicolin, ara-C and COMDP affect the elongation activity of DNA polymerase but not the initiation activity of the enzyme during DNA replication. This is the first report of such differentiation of the DNA polymerase activities during nuclear DNA replication in mammalian cells. The method of analysis described here for replication intermediates can be used to examine the inhibitory activities of other chemicals on DNA synthesis.     

A role for the common GTP-binding protein in coupling of chromosome replication to cell growth and cell division

Homologues of CgtA, the common GTP-binding protein of Vibrio harveyi, are present in diverse organisms ranging from bacteria to humans. In bacteria, proteins homologous to CgtA form a subfamily of small GTP-binding proteins, called Obg/Gtp1. Similarity between bacterial members of this subfamily and their eukaryotic homologues is as high as about 50%. Nevertheless, specific functions of these proteins remain largely unknown. Genes coding for CgtA-like proteins are essential in almost all species of bacteria. The only known exception is V. harveyi, whose cells survive disruption of the cgtA gene. Therefore, the V. harveyi cgtA insertional mutant is a very useful tool for studies on functions of CgtA. Here we demonstrate that under normal growth conditions, cells of the cgtA mutant are slightly larger than wild-type cells, whereas indirect inhibition of DNA replication initiation by addition of rifampicin results in significantly higher differences in average cell size between these two strains as measured by flow cytometry. These differences decreased when cell division was inhibited by cephalexin. DNA synthesis per cell mass was found to be increased in the cgtA mutant relative to wild-type V. harveyi strain, whereas the mutant cells grew slower than bacteria with functional cgtA gene. Kinetics of DNA replication after inhibition of cell division was also considerably different in wild-type and cgtA mutant strains. These results suggest that the cgtA gene product plays a role in coupling of DNA replication to cell growth and cell division.