Isolation and characterization of high-osmolarity-sensitive mutants of fission yeast

For the fission yeast Schizosaccharomyces pombe, adaptation to high-osmolarity medium is mediated by a mitogen-activated protein (MAP) kinase cascade, involving the Wis1 MAP kinase kinase and the Sty1 MAP kinase. The MAP kinase pathway transduces an osmotic signal and accordingly regulates the expression of the downstream target gene (gpd1(+)) that encodes NADH-dependent glycerol-3-phosphate dehydrogenase, in order to adaptively accumulate glycerol inside the cells as an osmoprotectant. We previously characterized a set of high-osmolarity-sensitive S. pombe mutants, including wis1, sty1, and gpd1. In this study, we attempted to further isolate novel osmolarity-sensitive mutants. For some of the mutants isolated, profiles of glycerol production in response to the osmolarity of the growth medium were indistinguishable from that of the wild-type cells, suggesting that they are novel types. They were classified into three distinct types genetically and, thus, were designated hos1, hos2, and hos3 (high osmolarity sensitive) mutants. One of them, the hos1 mutant, was characterized in detail. The hos1 mutant was demonstrated to have a mutational lesion in the known ryh1(+) gene, which encodes a small GTP-binding protein. Disruption of the ryh1(+) gene results not only in osmosensitivity but also in temperature sensitivity for growth. It was also found that the delta ryh1 mutant is severely sterile. These results are discussed with special reference to the osmoadaptation of S. pombe.     

Cut5 is a component of the UV-responsive DNA damage checkpoint in fission yeast

A checkpoint responding to DNA damage in G2 results in a delay in the onset of mitosis through inhibition of p34cdc2 kinase activity via maintenance of inhibitory tyrosine phosphorylation. Genetic analyses of this checkpoint in fission yeast have identified single alleles of several genes, suggesting these screens are not yet saturating, and hence further genes await identification. To fully understand the complexity of this checkpoint it will be necessary to define all the genes involved. To this end we screened for new mutants defective in the ability to delay mitosis in the presence of DNA-damaging agents. Twenty-four mutants were isolated that were defective in UV-C and MMS-induced checkpoint delay. Amongst these mutants was an allele of cut5 that was also defective in the checkpoint responses. We show here, contrary to previous reports, that the UV-C induced checkpoint response is defective in cut5 mutants. Therefore, like all other checkpoint mutants, cut5 is required for G2 checkpoint arrest following DNA damage, regardless of the nature of the lesions involved.     

A key role for replication factor C in DNA replication checkpoint function in fission yeast

Replication factor C (RF-C) is a five subunit DNA polymerase (Pol) delta/straightepsilon accessory factor required at the replication fork for loading the essential processivity factor PCNA onto the 3'-ends of nascent DNA strands. Here we describe the genetic analysis of the rfc2 +gene of the fission yeast Schizosaccharomyces pombe encoding a structural homologue of the budding yeast Rfc2p and human hRFC37 proteins. Deletion of the rfc2 + gene from the chromosome is lethal but does not result in the checkpoint-dependent cell cycle arrest seen in cells deleted for the gene encoding PCNA or for those genes encoding subunits of either Pol delta or Pol straightepsilon. Instead, rfc2 Delta cells proceed into mitosis with incompletely replicated DNA, indicating that the DNA replication checkpoint is inactive under these conditions. Taken together with recent results, these observations suggest a simple model in which assembly of the RF-C complex onto the 3'-end of the nascent RNA-DNA primer is the last step required for the establishment of a checkpoint-competent state.     

Identification of the ure1+ gene encoding urease in fission yeast

We describe a case of subacute cor pulmonale caused by tumor embolism from a gallbladder carcinoma in a 63-year-old woman. The patient was admitted to hospital with increasing dyspnea. Physical examination and echocardiography showed signs of pulmonary hypertension. She died of circulatory failure. At autopsy microscopic studies revealed tumor embolism in the pulmonary vessels and subsequent lesions causing the lethal pulmonary hypertension. This is the first case report of pulmonary hypertension caused by embolism from a gallbladder carcinoma in the literature worldwide.     

DNA damage checkpoint in budding yeast

Eukaryotic cells have evolved a network of control mechanisms, known as checkpoints, which coordinate cell-cycle progression in response to internal and external cues. The yeast Saccharomyces cerevisiae has been invaluable in dissecting genetically the DNA damage checkpoint pathway. Recent results on posttranslational modifications and protein-protein interactions of some key factors provide new insights into the architecture of checkpoint protein complexes and their order of function.     

Identification of Drosophila cytoskeletal proteins by induction of abnormal cell shape in fission yeast

To clone metazoan genes encoding regulators of cell shape, we have developed a functional assay for proteins that affect the morphology of a simple organism, the fission yeast Schizosaccharomyces pombe. A Drosophila melanogaster cDNA library was constructed in an inducible expression vector and transformed into S. pombe. When expression of the Drosophila sequences was induced, aberrant cell shapes were found in 0.2% of the transformed colonies. Four severe phenotypes representing defects in cytokinesis and/or cell shape maintenance were examined further. Each displayed drastic and specific reorganizations of the actin cytoskeleton. Three of the cDNAs responsible for these defects appear to encode cytoskeletal components: the actin binding proteins profilin and cofilin/actin depolymerizing factor and a membrane-cytoskeleton linker of the ezrin/merlin family. These results demonstrate that a yeast phenotypic screen efficiently identifies conserved genes from more complex organisms and sheds light on their potential in vivo functions.     

Identification of chlorobenzene dioxygenase sequence elements involved in dechlorination of 1,2,4,5-tetrachlorobenzene

The TecA chlorobenzene dioxygenase and the TodCBA toluene dioxygenase exhibit substantial sequence similarity yet have different substrate specificities. Escherichia coli cells producing recombinant TecA enzyme dioxygenate and simultaneously eliminate a halogen substituent from 1,2,4,5-tetrachlorobenzene but show no activity toward benzene, whereas those producing TodCBA dioxygenate benzene but not tetrachlorobenzene. A hybrid TecA dioxygenase variant containing the large alpha-subunit of the TodCBA dioxygenase exhibited a TodCBA dioxygenase specificity. Acquisition of dehalogenase activity was achieved by replacement of specific todC1 alpha-subunit subsequences by equivalent sequences of the tecA1 alpha-subunit. Substrate transformation specificities and rates by E. coli resting cells expressing hybrid systems were analyzed by high-performance liquid chromatography. This allowed the identification of both a single amino acid and potentially interacting regions required for dechlorination of tetrachlorobenzene. Hybrids with extended substrate ranges were generated that exhibited activity toward both benzene and tetrachlorobenzene. The regions determining substrate specificity in (chloro)benzene dioxygenases appear to be different from those previously identified in biphenyl dioxygenases.     

Identification of a putative alpha-glucan synthase essential for cell wall construction and morphogenesis in fission yeast

The cell wall protects fungi against lysis and determines their cell shape. Alpha-glucan is a major carbohydrate component of the fungal cell wall, but its function is unknown and its synthase has remained elusive. Here, we describe a fission yeast gene, ags1(+), which encodes a putative alpha-glucan synthase. In contrast to the structure of other carbohydrate polymer synthases, the predicted Ags1 protein consists of two probable catalytic domains for alpha-glucan assembly, namely an intracellular domain for alpha-glucan synthesis and an extracellular domain speculated to cross-link or remodel alpha-glucan. In addition, the predicted Ags1 protein contains a multipass transmembrane domain that might contribute to transport of alpha-glucan across the membrane. Loss of Ags1p function in a temperature-sensitive mutant results in cell lysis, whereas mutant cells grown at the semipermissive temperature contain decreased levels of cell wall alpha-glucan and fail to maintain rod shapes, causing rounding of the cells. These findings demonstrate that alpha-glucan is essential for fission yeast morphogenesis.     

Suppressors of cdc25p overexpression identify two pathways that influence the G2/M checkpoint in fission yeast

Checkpoints maintain the order of cell-cycle events. At G2/M, a checkpoint blocks mitosis in response to damaged or unreplicated DNA. There are significant differences in the checkpoint responses to damaged DNA and unreplicated DNA, although many of the same genes are involved in both responses. To identify new genes that function specifically in the DNA replication checkpoint pathway, we searched for high-copy suppressors of overproducer of Cdc25p (OPcdc25(+)), which lacks a DNA replication checkpoint. Two classes of suppressors were isolated. One class includes a new gene encoding a putative DEAD box helicase, suppressor of uncontrolled mitosis (sum3(+)). This gene negatively regulates the cell-cycle response to stress when overexpressed and restores the checkpoint response by a mechanism that is independent of Cdc2p tyrosine phosphorylation. The second class includes chk1(+) and the two Schizosaccharomyces pombe 14-3-3 genes, rad24(+) and rad25(+), which appear to suppress the checkpoint defect by inhibiting Cdc25p. We show that rad24Delta mutants are defective in the checkpoint response to the DNA replication inhibitor hydroxyurea at 37 degrees and that cds1Delta rad24Delta mutants, like cds1Delta chk1Delta mutants, are entirely checkpoint deficient at 29 degrees. These results suggest that chk1(+) and rad24(+) may function redundantly with cds1(+) in the checkpoint response to unreplicated DNA.     

HRAD1 and MRAD1 encode mammalian homologues of the fission yeast rad1(+) cell cycle checkpoint control gene

Eukaryotic cells arrest at the G2checkpoint in the presence of DNA damage or incompletely replicated DNA. This cell cycle checkpoint prevents the development and propagation of genomic instability. In the fission yeast, this process requires the action of a number of genes, including rad1(+) . We report here the identification of human and mouse cDNAs that exhibit extensive sequence homology to rad1(+) . The human gene, called HRAD1 , encodes a 282 amino acid protein that is 27% identical and 53% similar to yeast Rad1p. The human homologue maintains its sequence similarity over the full length of the protein, including the three proposed 3'-->5' exonuclease domains, and the leucine rich repeat region. The mouse gene, called MRAD1 , encodes a 280 amino acid protein that is 90% identical and 96% similar to HRAD1 at the amino acid level. Expression of HRAD1 in yeast rad1 mutants partially restores radiation resistance and G2checkpoint proficiency to these mutants. Evolutionaryconservation of structure between HRAD1 , MRAD1 , rad1(+), Saccharomyces cerevisiae RAD17 and the Ustilago maydis REC1 checkpoint genes suggests that the function of the encoded proteins is conserved as well. The ability of HRAD1 to partially complement yeast rad1 mutants suggests that this gene is required for G2checkpoint control in human cells.     

S-phase DNA damage checkpoint in budding yeast

Eukaryotic cells must be able to coordinate DNA repair, replication and cell cycle progression in response to DNA damage. A failure to activate the checkpoints which delay the cell cycle in response to internal and external cues and to repair the DNA lesions results in an increase in genetic instability and cancer predisposition. The use of the yeast Saccharomyces cerevisiae has been invaluable in isolating many of the genes required for the DNA damage response, although the molecular mechanisms which couple this regulatory pathway to different DNA transactions are still largely unknown. In analogy with prokaryotes, we propose that DNA strand breaks, caused by genotoxic agents or by replication-related lesions, trigger a replication coupled repair mechanism, dependent upon recombination, which is induced by the checkpoint acting during S-phase.     

Lineage-dependent mating-type transposition in fission and budding yeast

The basis of cellular differentiation is perhaps best understood in the yeast mating-type switching system. The yeast cell produces daughter cells that differ from each other or from their parent cell via developmentally regulated genomic rearrangements. Recent experiments on cell-type determination in fission yeast have revealed that this process is determined by the inheritance of specific parental chromosome strands by the progeny cells.     

Identification of fission yeast nuclear markers using random polypeptide fusions with green fluorescent protein

We describe a method for identifying genes encoding proteins with stereospecific intracellular localizations in the fission yeast Schizosaccharomyces pombe. Yeast are transformed with a gene library in which S. pombe genomic sequences are fused to the gene encoding the Aequorea victoria green fluorescent protein (GFP), and intracellular localizations are subsequently identified by rapid fluorescence screening in vivo. In a model application of these methods to the fission yeast nucleus, we have identified several novel genes whose products are found in specific nuclear regions, including chromatin, the nucleolus, and the mitotic spindle, and sequence similarities between some of these genes and previously identified genes encoding nuclear proteins have validated the approach. These methods will be useful in identifying additional components of the S. pombe nucleus, and further extensions of this approach should also be applicable to a more comprehensive identification of the elements of intracellular architecture in fission yeast.     

Identification and characterization of srp1, a gene of fission yeast encoding a RNA binding domain and a RS domain typical of SR splicing factors

Smad6 and Smad7 function as intracellular antagonists in transforming growth factor-beta (TGF-beta) signaling. Here we report the isolation of human Smad6, which is closely related to Smad7. Smad6 and Smad7 mRNAs were differentially expressed in lung cancer cell lines and were rapidly and directly induced by TGF-beta1, activin and bone morphogenetic protein-7. Cross-talk between TGF-beta and other signaling pathways was demonstrated by the finding that epidermal growth factor (EGF) induced the expression of inhibitory SMAD mRNA. Moreover, whereas the phorbol ester PMA alone had no effect, it potentiated the TGF-beta1-induced expression of Smad7 mRNA. Ectopic expression of anti-sense Smad7 RNA was found to increase the effect of TGF-beta1, supporting its role as a negative regulator in TGF-beta signaling. Thus, expression of inhibitory Smads is induced by multiple stimuli, including the various TGF-beta family members, whose action they antagonize.