Specificity of replicating instability in Schizosaccharomyces pombe haploid yeasts

UV-induced genetic instability in haploid Schizosaccharomyces pombe does not appear to be very locus-specific. This conclusion contradicts the data previously published by other authors. The possible causes for this discrepancy are discussed.     

The evolution of transposons in Schizosaccharomyces pombe

Recent studies of the LTR-retrotransposons of Schizosaccharomyces pombe have shed considerable light on their evolution and function. The sequencing of the S. pombe genome allowed analysis of its transposon content. This analysis provides information about the maintenance and loss of transposons in the genome. The results of transposition assays and biochemical analyses demonstrate that the N-terminal protein of Tf1 is functionally equivalent to the Gag proteins of retroviruses and retrotransposons. Despite this conservation of function, the N-terminal protein of Tf1 lacks any sequence similarity to other known Gag proteins. Sequence analysis and experimental data also indicate that the Tf1 transposons of S. pombe target their integration into specific sites in the host genome. Transposition events resulting from the expression of Tf1 reveal a strong preference for intergenic regions, specifically at pol II promoters in a window 100-400 bp upstream of open reading frames. The complete and partial copies of Tf transposons in the sequenced genome of S. pombe show the same association of integration with promoter regions. This body of work explores how the transposon interacts with the host, the balance between the transposons propagation and loss, and how different families of transposons evolve.     

Identification of 24-methylene-24,25-dihydrolanosterol as a precursor of ergosterol in the yeasts Schizosaccharomyces pombe and Schizosaccharomyces octosporus

Abstract Study of the plasma membrane sterol composition in the yeasts Schizosaccharomyces pombe and Schizosaccharomyces octosporus revealed the presence of ergosterol, lanosterol, dehydroergosterol, fecosterol, episterol and 24-methylene-24,25-dihydrolanosterol (eburicol), a C-31 derivative. The growth of both yeasts in the presence of ketoconazole led to a decrease by 85% of the ergosterol content while the levels of lanosterol and eburicol increased. This suggests that in the biosynthetic pathway of ergosterol in Schizosaccharomyces species, the transmethylation process on the C-24 may occur directly on lanosterol and not only on zymosterol. On the other hand, it cannot be excluded that in the genus Schizosaccharomyces two routes exist from lanosterol to ergosterol: the classical one via a direct C-14, C-4 demethylation of lanosterol and the second one via the formation of a C-31 derivative followed by demethylations.     

A study of the maloalcoholic fermentation pathway in Schizosaccharomyces pombe.

The pathway of the maloalcoholic fermentation in Schizosaccharomyces pombe was investigated by a 1H-, 2H- and 13C-n.m.r.-spectroscopic study of hydrogen and deuterium distribution on the ethanol produced by S. pombe from L-malic acid in 2H2O and from L-[2-2H]malic acid. Our findings rule out a double-decarboxylation mechanism and agree with a pathway that involves acetaldehyde as intermediate.     

Microtubule-driven nuclear movements and linear elements as meiosis-specific characteristics of the fission yeasts Schizosaccharomyces versatilis and Schizosaccharomyces pombe

Meiotic prophase in Schizosaccharomyces pombe is characterized by striking nuclear movements and the formation of linear elements along chromosomes instead of tripartite synaptonemal complexes. We analysed the organization of nuclei and microtubules in cells of fission yeasts undergoing sexual differentiation. S. japonicus var. versatilis and S. pombe cells were studied in parallel, taking advantage of the better cytology in S. versatilis. During conjugation, microtubules were directed towards the mating projection. These microtubules seem to lead the haploid nuclei together in the zygote by interaction with the spindle pole bodies at the nuclear periphery. After karyogamy, arrays of microtubules emanating from the spindle pole body of the diploid nucleus extended to both cell poles. The same differentiated microtubule configuration was elaborated upon induction of azygotic meiosis in S. pombe. The cyclic movements of the elongated nuclei between the cell poles is reflected by a dynamic and coordinated shortening and lengthening of the two microtubule arrays. When the nucleus was at a cell end, one array was short while the other bridged the whole cell length. Experiments with inhibitors showed that microtubules are required for karyogamy and for the elongated shape and movement of nuclei during meiotic prophase. In both fission yeasts the SPBs and nucleoli are at the leading ends of the moving nuclei. Astral and cytoplasmic microtubules were also prominent during meiotic divisions and sporulation. We further show that in S. versatilis the linear elements formed during meiotic prophase are similar to those in S. pombe. Tripartite synaptonemal complexes were never detected. Taken together, these findings suggest that S. pombe and S. versatilis share basic characteristics in the organization of microtubules and the structure and behaviour of nuclei during their meiotic cell cycle. The prominent differentiations of microtubules and nuclei may be involved in the pairing, recombination, and segregation of meiotic chromosomes.     

Interaction of Epe1 with the heterochromatin assembly pathway in Schizosaccharomyces pombe

Epe1 is a JmjC domain protein that antagonizes heterochromatization in Schizosaccharomyces pombe. Related JmjC domain proteins catalyze a histone demethylation reaction that depends on Fe(II) and alpha-ketoglutarate. However, no detectable demethylase activity is associated with Epe1, and its JmjC domain lacks conservation of Fe(II)-binding residues. We report that Swi6 recruits Epe1 to heterochromatin and that overexpression of epe1+, like mutations in silencing genes or overexpression of swi6+, upregulates expression of certain genes. A significant overlap was observed between the lists of genes that are upregulated by overexpression of epe1+ and those that are upregulated by mutations in histone deacetylase genes. However, most of the common genes are not regulated by Clr4 histone methyltransferase. This suggests that Epe1 interacts with the heterochromatin assembly pathway at the stage of histone deacetylation. Mutational inactivation of Epe1 downregulates approximately 12% of S. pombe genes, and the list of these genes overlaps significantly with the lists of genes that are upregulated by mutations in silencing genes and genes that are hyperacetylated at their promoter regions in clr6-1 mutants. We propose that an interplay between the repressive HDACs activity and Epe1 helps to regulate gene expression in S. pombe.     

In vitro reconstitution of the Schizosaccharomyces pombe alternative excision repair pathway

Schizosaccharomyces pombe alternative excision repair has been shown genetically and biochemically to be involved in the repair of a wide variety of DNA lesions. AER is initiated by a damage-specific endonuclease (Uve1p) that recognizes UV-induced photoproducts, base mispairs, abasic sites, and platinum G-G diadducts and cleaves the DNA phosphodiester backbone 5' to a lesion. Several models exist that employ various mechanisms for damage removal based on the activities of Rad2p, a nuclease thought to be responsible for damage excision in AER. This study represents the first report of the biochemical reconstitution of the AER pathway. A base mispair-containing substrate is repaired in a reaction requiring S. pombe Uve1p, Rad2p, DNA polymerase delta, replication factor C, proliferating cell nuclear antigen, and T4 DNA ligase. Surprisingly, damage is removed exclusively by the 5' to 3' exonuclease activity of Rad2p and not its "flap endonuclease" activity and is absolutely dependent upon the presence of the 5'-phosphoryl moiety at the Uve1p cleavage site.     

A comparative analysis of an orthologous proteomic environment in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe

The sequential application of protein tagging, affinity purification, and mass spectrometry enables highly accurate charting of proteomic environments by the characterization of stable protein assemblies and the identification of subunits that are shared between two or more protein complexes, termed here "proteomic hyperlinks." We have charted the proteomic environments surrounding the histone methyltransferase, Set1, in both yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Although the composition of these nonessential Set1 complexes is remarkably conserved, they differ with respect to their hyperlinks to their proteomic environments. We speculate that conservation of the core components of protein assemblies and variability of hyperlinks represents a general principle in the molecular organization of eukaryotic proteomes.     

Expression of the ADP/ATP carrier encoding genes in aerobic yeasts; phenotype of an ADP/ATP carrier deletion mutant of Schizosaccharomyces pombe

The expression of a key mitochondrial membrane component, the ADP/ATP carrier, was investigated in two aerobic yeast species, Kluyveromyces lactis and Schizosaccharomyces pombe. Although the two species differ very much in their respiratory capacity, the expression of the carrier in both yeast species was decreased under partially anaerobic conditions and was induced by nonfermentable carbon sources. The single ADP/ATP carrier encoding gene was deleted in S. pombe. The null mutant exhibits impaired growth properties, especially when cultivated at reduced oxygen tension, and is unable to grow on a nonfermentable carbon source. Our results suggest that the inability of K. lactis and S. pombe to grow under anaerobic conditions can be related in part to the absence of a functional ADP/ATP carrier due to repression of the corresponding gene expression.     

Expression of a VEGF-like protein from Parapoxvirus ovis in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe

We report on the expression of a VEGF-like protein encoded by Parapoxvirus ovis in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. We show that a lysine residue at amino acid position 2 (K2) is an important determinant for the stability of this protein in S. cerevisiae. Replacement of K2 by an arginine results in stabilization of the protein. This observation suggests that this lysine may be a target for ubiquitinylation, which is a prerequisite for proteasome-mediated protein degradation. Interestingly, in S. pombe the lysine (K2) has no influence on the stability of the protein. This result indicates that the two yeast species exhibit significant differences in their protein degradation pathways.     

Function of the ypt2 gene in the exocytic pathway of Schizosaccharomyces pombe.

The ypt2 gene of the fission yeast Schizosaccharomyces pombe encodes a member of the ypt/rab family of small GTP-binding proteins, related in sequence to Sec4p of Saccharomyces cerevisiae but closer to mammalian rab8. We have introduced a mutation into the gene corresponding to a mutation identified in ypt1, in which a conserved valine residue was altered to asparagine. The mutated ypt2 gene was introduced into the S. pombe genome by gene replacement. The resulting strain was temperature-sensitive for growth. Normal growth was restored by introduction of a plasmid-borne wild-type ypt2 cDNA or by cDNA for rab8 but not by various other rab or ypt sequences. At restrictive temperature the mutant cells accumulated the secretory protein acid phosphatase in a form that appeared to be fully glycosylated and acquired a population of vesicles detectable by electron microscopy. Thus the ypt2 protein, and by inference rab8, appear to function in the last stage of the secretory pathway.     

Simulating the evolution of signal transduction pathways

We use a generic model of a network of proteins that can activate or deactivate each other to explore the emergence and evolution of signal transduction networks and to gain a basic understanding of their general properties. Starting with a set of non-interacting proteins, we evolve a signal transduction network by random mutation and selection to fulfill a complex biological task. In order to validate this approach we base selection on a fitness function that captures the essential features of chemotactic behavior as seen in bacteria. We find that a system of as few as three proteins can evolve into a network mediating chemotaxis-like behavior by acting as a "derivative sensor". Furthermore, we find that the dynamics and topology of such networks show many similarities to the natural chemotaxis pathway, that the response magnitude can increase with increasing network size and that network behavior shows robustness towards variations in some of the internal parameters. We conclude that simulating the evolution of signal transduction networks to mediate a certain behavior may be a promising approach for understanding the general properties of the natural pathway for that behavior.     

Characterization of the alternative excision repair pathway of UV-damaged DNA in Schizosaccharomyces pombe.

Schizosaccharomyces pombe cells deficient in nucleotide excision repair (NER) are still able to remove photoproducts from cellular DNA, showing that there is a second pathway for repair of UV damage in this organism. We have characterized this repair pathway by cloning and disruption of the genomic gene encoding UV damage endonuclease (UVDE). Although uvde gene disruptant cells are only mildly UV sensitive, a double disruptant of uvde and rad13 (a S. pombe mutant defective in NER) was synergistically more sensitive than either single disruptant and was unable to remove any photoproducts from cellular DNA. Analysis of the kinetics of photoproduct removal in different mutants showed that the UVDE-mediated pathway operates much more rapidly than NER. In contrast to a previous report, our genetic analysis showed that rad12 and uvde are not the same gene. Disruption of the rad2 gene encoding a structure- specific flap endonuclease makes cells UV sensitive, but much of this sensitivity is not observed if the uvde gene is also disrupted. Further genetic and immunochemical analyses suggest that DNA incised by UVDE is processed by two separate mechanisms, one dependent and one independent of flap endonuclease.