The Schizosaccharomyces pombe spo3+ gene is required for assembly of the forespore membrane and genetically interacts with psy1(+)-encoding syntaxin-like protein.

Formation of the forespore membrane, which becomes the plasma membrane of spores, is an intriguing step in the sporulation of the fission yeast Schizosaccharomyces pombe. Here we report two novel proteins that localize to the forespore membrane. spo3(+) encodes a potential membrane protein, which was expressed only during sporulation. Green fluorescent protein (GFP) fusion revealed that Spo3 localized to the forespore membrane. The spo3 disruptant was viable and executed meiotic nuclear divisions as efficiently as the wild type but did not form spores. One of the spo3 alleles, spo3-KC51, was dose-dependently suppressed by psy1(+), which encodes a protein similar to mammalian syntaxin-1A, a component of the plasma membrane docking/fusion complex. psy1(+) was essential for vegetative growth, and its transcription was enhanced during sporulation. As expected, Psy1 localized to the plasma membrane during vegetative growth. Interestingly, Psy1 on the plasma membrane disappeared immediately after first meiotic division and relocalized to the forespore membrane as the second division initiated. In the spo3 null mutant, the forespore membrane was initiated but failed to develop a normal morphology. Electron microscopy revealed that membrane vesicles were accumulated in the cytoplasm of immature spo3Delta asci. These results suggest that Spo3 is a key component of the forespore membrane and is essential for its assembly acting in collaboration with the syntaxin-like protein.

The 5' terminal region of the Schizosaccharomyces pombe mes1 mRNA is crucial for its meiosis-specific splicing.

The mes1+ gene of Schizosaccharomyces pombe is required for the second meiotic division. The single 75-nt intron in mes1 is spliced out only in meiotic cells. Here we report a cis-acting element which is responsible for meiosis-specific splicing. Both 5' and 3' splice sites of the mes1 intron deviate from the consensus sequence. Point mutations which altered these sites so that they conformed to the consensus, however, did not affect the splicing pattern of mes1. Neither replacement of the mes1 intron with the constitutively spliced intron of the nda3 gene, nor replacement of the 3' exon with E. coli lacZ changed the splicing pattern. In contrast, deletion of the 5' terminal 125 nt from the 5' exon derepressed splicing in vegetative cells, implying that this 5' terminal sequence, named SRE (mes1 splicing repression element), inhibits splicing of the downstream intron. A potential stem-loop structure in the SRE is predicted. Disruption of this stem structure by mutation abolished the repression of mes1 splicing in vegetative cells. Overexpression of the SRE sequence on a multicopy plasmid also relieved the repression of splicing of the authentic mes1 transcripts. These results suggest that as yet unknown trans-acting factors inhibit splicing of the mes1 transcript in vegetative cells by interacting with the cis-element SRE.

A novel Cdc20-related WD-repeat protein, Fzr1, is required for spore formation in Schizosaccharomyces pombe.

Ste9/Srw1 which shows sequence homology to Hctl from budding yeast, is an activator of the anaphase-promoting complex (APC) in the fission yeast Schizosaccharomyces pombe. By homology search of the S. pombe genome, we identified the gene fr1+, which encodes the protein with the highest homology to Ste9 among five Cdc20-like proteins. Like Ste9, Fzr1 contains seven WD-repeats in its C-terminal region. In spite of this structural similarity, however, overproduction of either of these proteins cannot complement mutants lacking the other. fzr1+ is transcribed exclusively during meiosis and sporulation, suggesting that it plays a role in these processes. In fact, the fzr1 disruptant formed aberrant asci, which contained only one or two mature spores, though meiotic nuclear divisions proceeded with kinetics similar to wild type, and meiotic segregation of chromosomes was normal. Structural alteration of spindle pole bodies, which is a prerequisite for the formation of the forespore membrane, occurred normally in fzr1delta during the second meiotic division. Localization of spore rim marker proteins fused to green fluorescent protein showed that nascent prespores were irregularly shaped, small in size and few in number in fzr1delta cells compared to wild-type cells. Furthermore, electron microscopy revealed that the outer layer of the spore walls was often missing in fzr1delta spores. These results show that Fzr1 is specifically involved in the assembly of the spore envelope and also in spore maturation. Fzr1, a structural homolog of the APC regulator, therefore plays an important role in spore morphogenesis.

Comprehensive isolation of meiosis-specific genes identifies novel proteins and unusual non-coding transcripts in Schizosaccharomyces pombe.

In order to isolate meiosis-specific genes in Schizosaccharomyces pombe, we have constructed a subtracted cDNA library enriched in clones whose expression is enhanced during meiosis induced by nitrogen starvation. Using northern blot analysis, we isolated 31 kinds of clones whose expression was induced in a meiosis/sporulation-specific manner. We comprehensively named them meu after meiotic expression upregulated. The transcription of 20 meu genes was found to be dependent on the mei4(+) gene, which encodes a transcription factor required for the progression of meiosis. DNA sequencing indicated that most of the meu genes encode novel proteins. Notably, five of the meu genes harbor no apparent protein coding sequences, and the transcripts form stable hairpin structures, suggesting that they may generate non-coding RNAs or antisense RNAS: The results presented here imply that RNAs are also important for the comprehensive characterization of genomic expression.

The Schizosaccharomyces pombe spo20(+) gene encoding a homologue of Saccharomyces cerevisiae Sec14 plays an important role in forespore membrane formation.

The Schizosaccharomyces pombe spo20-KC104 mutation was originally isolated in a screen for sporulation-deficient mutants, and the spo20-KC104 mutant exhibits temperature-sensitive growth. Herein, we report that S. pombe, spo20(+) is essential for fission yeast cell viability and is constitutively expressed throughout the life cycle. We also demonstrate that the spo20(+) gene product is structurally homologous to Saccharomyces cerevisiae Sec14, the major phosphatidylinositol transfer protein of budding yeast. This structural homology translates to a significant degree of functional relatedness because reciprocal complementation experiments demonstrate that each protein is able to fulfill the essential function of the other. Moreover, biochemical experiments show that, like Sec14, Spo20 is a phosphatidylinositol/phosphatidylcholine-transfer protein. That Spo20 is required for Golgi secretory function in vegetative cells is indicated by our demonstration that the spo20-KC104 mutant accumulates aberrant Golgi cisternae at restrictive temperatures. However, a second phenotype observed in Spo20-deficient fission yeast is arrest of cell division before completion of cell separation. Consistent with a direct role for Spo20 in controlling cell septation in vegetatively growing cells, localization experiments reveal that Spo20 preferentially localizes to the cell poles and to sites of septation of fission yeast cells. We also report that, when fission yeasts are challenged with nitrogen starvation, Spo20 translocates to the nucleus. This nuclear localization persists during conjugation and meiosis. On completion of meiosis, Spo20 translocates to forespore membranes, and it is the assembly of forespore membranes that is abnormal in spo20-KC104 cells. In such mutants, a considerable fraction of forming prespores fail to encapsulate the haploid nucleus. Our results indicate that Spo20 regulates the formation of specialized membrane structures in addition to its recognized role in regulating Golgi secretory function.

The cyclic AMP/PKA signal pathway is required for initiation of spore germination in Schizosaccharomyces pombe.

Spore germination, a transition from the quiescent G0 phase to the proliferation cycle, is triggered by glucose in Schizosaccharomyces pombe. The role of cAMP/protein kinase A (PKA) signalling in germination is investigated. Gene disruption of cyr1+, pka1+ and gpa2+ encoding adenylate cyclase, PKA and the alpha-subunit of a trimeric GTP-binding protein, respectively, reduced the colony-forming efficiency of spores in minimal medium. Isolated spores of these null mutants did not germinate in minimal medium for up to 12 h, at which time wild-type spores had completed germination and formed germ projections. In wild-type spores, cortical actin patches randomly distributed in the early stage of outgrowth and then localized to one side of spores before the formation of projections. In contrast, the mutant spores exhibited no actin patches, but the cell surface was predominantly stained, like ungerminated spores of wild-type. Flow fluorocytometric analysis of propidium iodide-stained spores revealed a distinct 1C DNA peak after germination was completed. The fluorescent profile of the mutant spores, however, did not change during 12 h incubation in the minimal medium. These observations indicate that spores harbouring either cyr1Delta, pka1Delta or gpa2Delta are hardly triggered to germination. When wild-type spores were exposed to glucose, the intracellular cAMP level transiently increased in a few minutes, but gpa2Delta spores did not respond to glucose. We conclude that S. pombe spores initiate germination in response to glucose through the cyclic AMP-PKA pathway.

The Schizosaccharomyces pombe spo6+ gene encoding a nuclear protein with sequence similarity to budding yeast Dbf4 is required for meiotic second division and sporulation.

BACKGROUND: Sporulation of the fission yeast Schizosaccharomyces pombe is a cell differentiation process which accompanies meiosis. The spo6+ gene was identified as a sporulation-specific gene, whose transcription was regulated by the forkhead family transcription factor Mei4. RESULTS: spo6+ encodes a protein with sequence similarity to Saccharomyces cerevisiae Dbf4p, which is required for the initiation of DNA replication. However, doubling time and cell morphology of spo6 deletion mutants and spo6-cDNA over-expressing cells were indistinguishable from wild-type cells. Spliced mature mRNAs of spo6+ appeared when diploid cells committed to meiosis. Spo6p fused to green fluorescent protein (GFP) preferentially localized in a nucleus. Although spo6Delta diploids normally underwent premeiotic DNA replication and meiosis-I, approximately 80% of cells were blocked at the binucleate stage during meiosis and virtually no asci were formed. Anti-tubulin staining revealed that only 25% of the binucleate cells assembled spindle microtubules for meiosis-II. In a small number of tetranucleate cells, sister nuclei insufficiently separated and spindles were frequently fragmented. The meiosis-II arrest phenotype was exaggerated at low temperature and in the presence of caffeine. CONCLUSIONS: These results indicate that Spo6p is a novel Dbf4-related nuclear protein, which is expressed during meiosis and is indispensable for normal progression of meiosis-II and sporulation.

Positioning of medial actin rings affected by eccentrically located nuclei in a fission yeast mutant having large vacuoles.

In Schizosaccharomyces pombe wild-type cells, the nucleus positions in the middle of the cell where the cortical actin ring is assembled prior to septum formation. ste12 mutants contain a few large vacuoles. In a considerable fraction of ste12 cells, the nuclei and septa were eccentrically positioned. Both extension of spindle microtubules in the anaphase and post-mitotic migration of sister nuclei to the cell poles were partially disrupted, probably due to enlarged vacuoles. In spite of the eccentric positioning of nuclei, the cortical actin ring overlays the displaced pre-mitotic nucleus. This observation supports the notion that the nucleus dictates the division site in fission yeast.

Autoregulated expression of Schizosaccharomyces pombe meiosis-specific transcription factor Mei4 and a genome-wide search for its target genes.

The Schizosaccharomyces pombe mei4(+) gene encoding a forkhead transcription factor is necessary for the progression of meiosis and sporulation. We searched for novel meiotic genes, the expression of which is dependent on Mei4p, since only the spo6(+) gene has been assigned to its targets. Six known genes responsible for meiotic recombination were examined by Northern blotting, but none were Mei4 dependent for transcription. We determined the important cis-acting element, designated FLEX, to which Mei4p can bind. The S. pombe genome sequence database (The Sanger Centre, UK) was scanned for the central core heptamer and its flanking 3' sequence of FLEX composed of 17 nucleotides, and 10 candidate targets of Mei4 were selected. These contained a FLEX-like sequence in the 5' upstream nontranslatable region within 1 kb of the initiation codon. Northern blotting confirmed that 9 of them, named mde1(+) to mde9(+), were transcriptionally induced during meiosis and were dependent on mei4(+). Most mde genes have not been genetically defined yet, except for mde9(+), which is identical to spn5(+), which encodes one of the septin family of proteins. mde3(+) and a related gene pit1(+) encode proteins related to Saccharomyces cerevisiae Ime2. The double disruptant frequently produced asci having an abnormal number and size of spores, although it completed meiosis. We also found that the forkhead DNA-binding domain of Mei4p binds to the FLEX-like element in the putative promoter region of mei4 and that the maximum induction level of mei4 mRNA required functional mei4 activity. Furthermore, expression of a reporter gene driven by the authentic mei4 promoter was induced in vegetative cells by ectopic overproduction of Mei4p. These results suggest that mei4 transcription is positively autoregulated.

S. pombe sporulation-specific coiled-coil protein Spo15p is localized to the spindle pole body and essential for its modification.

Spindle pole bodies in the fission yeast Schizosaccharomyces pombe are required during meiosis, not only for spindle formation but also for the assembly of forespore membranes. The spo15 mutant is defective in the formation of forespore membranes, which develop into spore envelopes. The spo15(+)gene encodes a protein with a predicted molecular mass of 223 kDa, containing potential coiled-coil regions. The spo15 gene disruptant was not lethal, but was defective in spore formation. Northern and western analyses indicated that spo15(+) was expressed not only in meiotic cells but also in vegetative cells. When the spo15-GFP fusion gene was expressed by the authentic spo15 promoter during vegetative growth and sporulation, the fusion protein colocalized with Sad1p, which is a component of spindle pole bodies. Meiotic divisions proceeded in spo15delta cells with kinetics similar to those in wild-type cells. In addition, the morphology of the mitotic and meiotic spindles and the nuclear segregation were normal in spo15delta. Intriguingly, transformation of spindle pole bodies from a punctate to a crescent form prior to forespore membrane formation was not observed in spo15delta cells. We conclude that Spo15p is associated with spindle pole bodies throughout the life cycle and plays an indispensable role in the initiation of spore membrane formation.

Replication factor C3 of Schizosaccharomyces pombe, a small subunit of replication factor C complex, plays a role in both replication and damage checkpoints.

We report here the isolation and functional analysis of the rfc3(+) gene of Schizosaccharomyces pombe, which encodes the third subunit of replication factor C (RFC3). Because the rfc3(+) gene was essential for growth, we isolated temperature-sensitive mutants. One of the mutants, rfc3-1, showed aberrant mitosis with fragmented or unevenly separated chromosomes at the restrictive temperature. In this mutant protein, arginine 216 was replaced by tryptophan. Pulsed-field gel electrophoresis suggested that rfc3-1 cells had defects in DNA replication. rfc3-1 cells were sensitive to hydroxyurea, methanesulfonate (MMS), and gamma and UV irradiation even at the permissive temperature, and the viabilities after these treatments were decreased. Using cells synchronized in early G2 by centrifugal elutriation, we found that the replication checkpoint triggered by hydroxyurea and the DNA damage checkpoint caused by MMS and gamma irradiation were impaired in rfc3-1 cells. Association of Rfc3 and Rad17 in vivo and a significant reduction of the phosphorylated form of Chk1 in rfc3-1 cells after treatments with MMS and gamma or UV irradiation suggested that the checkpoint signal emitted by Rfc3 is linked to the downstream checkpoint machinery via Rad17 and Chk1. From these results, we conclude that rfc3(+) is required not only for DNA replication but also for replication and damage checkpoint controls, probably functioning as a checkpoint sensor.

Fission yeast Ste9, a homolog of Hct1/Cdh1 and Fizzy-related, is a novel negative regulator of cell cycle progression during G1-phase.

When proliferating fission yeast cells are exposed to nitrogen starvation, they initiate conjugation and differentiate into ascospores. Cell cycle arrest in the G1-phase is one of the prerequisites for cell differentiation, because conjugation occurs only in the pre-Start G1-phase. The role of ste9(+) in the cell cycle progression was investigated. Ste9 is a WD-repeat protein that is highly homologous to Hct1/Cdh1 and Fizzy-related. The ste9 mutants were sterile because they were defective in cell cycle arrest in the G1-phase upon starvation. Sterility was partially suppressed by the mutation in cig2 that encoded the major G1/S cyclin. Although cells lacking Ste9 function grow normally, the ste9 mutation was synthetically lethal with the wee1 mutation. In the double mutants of ste9 cdc10(ts), cells arrested in G1-phase at the restrictive temperature, but the level of mitotic cyclin (Cdc13) did not decrease. In these cells, abortive mitosis occurred from the pre-Start G1-phase. Overexpression of Ste9 decreased the Cdc13 protein level and the H1-histone kinase activity. In these cells, mitosis was inhibited and an extra round of DNA replication occurred. Ste9 regulates G1 progression possibly by controlling the amount of the mitotic cyclin in the G1-phase.

The Schizosaccharomyces pombe mei4+ gene encodes a meiosis-specific transcription factor containing a forkhead DNA-binding domain.

The mei4+ gene of the fission yeast Schizosaccharomyces pombe was cloned by functional complementation. The mei4 disruptant failed to complete meiosis-I but could proliferate normally. mei4+ was transcribed only in meiosis-proficient diploid cells after premeiotic DNA replication. The mei4+ open reading frame encodes a 57-kDa serine-rich protein comprised of 517 amino acids with a forkhead/HNF3 DNA-binding domain in the amino-terminal region. Transcription of spo6+, a gene required for sporulation, was dependent on the mei4+ function. Two copies of the GTAAAYA consensus sequence, proposed as the binding site for human forkhead proteins, were found in the promoter region of spo6+. A gel mobility shift assay demonstrated the sequence-dependent binding of the GST-Mei4 forkhead domain fusion protein to DNA fragments with one of the consensus elements. Deletion of this consensus element from the spo6 promoter abolished the transcription of spo6+ and resulted in a sporulation deficiency. One-hybrid assay of Mei4 which was fused to the Gal4 DNA-binding domain localized the transcriptional activation domain in the C-terminal 140 amino acids of Mei4. These results indicate that Mei4 functions as a meiosis-specific transcription factor of S. pombe.

The Ste16 WD-repeat protein regulates cell-cycle progression under starvation through the Rum1 protein in Schizosaccharomyces pombe.

The haploid cells of the fission yeast, Schizosaccharomyces pombe, are arrested in the G1-phase by nitrogen starvation and are committed to sexual reproduction (mating and sporulation). We isolated the sterile mutants which were defective in G1 arrest following nitrogen starvation. Genetic analysis of these mutants defined a single locus designated as ste16. The nucleotide sequence revealed that ste16+ encodes an 82-kDa protein containing eight WD40-repeats in its carboxy terminal half. The ste16 disruptant was viable, but arrested the cell cycle in the G2-phase after the nutritional down-shift. When transferred to fresh growth medium, the G2-arrested ste16Delta haploids resumed the mitotic cycle from the S-phase, resulting in diploidization. This diploidization phenomenon was completely suppressed by the null mutation of rum1 encoding the inhibitor of Cdc2 kinase. As the Rum1 protein level was remarkably elevated in the ste16Delta, the Ste16 protein negatively controls the Rum1 level. The loss of function of ste16 disturbs the cell-cycle progression and impairs the mechanism for the maintenance of ploidy.

[Electrophysiologic evaluation of passive and active attentions--I. Topographic analysis of somatosensory event-related potentials].

We studied the topography of somatosensory event-related potentials (SERP) in two different attentive conditions: passive and active. Seventeen healthy right-handed young men, aged 19 to 28 years old (average age; 22.9 years), were requested to perform the following four paradigms in turn. In the passive attentive paradigm, participants were given no specific task when 0.3 Hz electrical stimuli were at random delivered to the right median nerve. In the active attentive paradigm, subjects were required to direct their attention to the regular 0.3 Hz stimuli with all their might. In the control paradigm. 2.0 Hz stimuli were delivered as they listened to their favorite music. Finally, in the oddball paradigm, subjects were instructed to push a button whenever they detected rare stimuli. SERP was recorded at 13 electrodes Grand average topographic amplitude maps from the individual data of the seventeen subjects were made at the latencies of N 60, P 90, N 130 and P 250 for each of the four paradigms. The topographic maps of P 250, the largest positive peak between 200 and 300 msec after the stimuli, showed a significant difference in the distribution of amplitude in the passive and active attentive paradigms compared to the control paradigm. A statistically significant enhancement was noted at the central region in the passive attention, and at the central and frontal regions in the active attention. These results suggest that P 250 consists of two attentive components distinct from selective or discriminative attention. The enhanced positivity at the frontal region during active attention may be associated with expecting the next stimulus and maintaining awareness.

[Electrophysiologic evaluation of passive and active attentions--II. Developmental changes of somatosensory event-related potentials in different attentive paradigms].

By somatosensory event-related potentials (SERP) we have previously demonstrated in adults that an enhanced positivity of P 250 is statistically significant at the frontal region during the active attentive state. We have also evaluated developmental changes of P 250 during different attentive states: passive and active. In this study, 30 healthy children (5-16 years of age) and six healthy adults (23-41 years) were required to perform the following four paradigms: passive attentive, active attentive, control, and oddball. SERP were recorded at Fz, Cz and Pz. In the passive attentive paradigm, P250 amplitude at Fz, Cz and Pz remained constant irrespective of age. With regard to the topography of the amplitude, however, P 250 was distributed dominantly at Pz in Group 1 (5-6 years of age) and Group 2 (7-11 years), contrary to the dominance at Cz in both Group 3 (12-16 years) and adults. In the active attentive paradigm, on the other hand, the P 250 amplitude at Fz reached its minimum at about 10 years and then increased until the adulthood. With respect to the topography of the amplitude, P 250 was distributed dominantly at Pz in Groups 1 and 2, at Cz in Group 3, and at Fz and Cz in adults. These results suggest that the topographic changes in the active attentive paradigm are dependent upon the developing abilities to expect the next stimulus and to maintain awareness.

Functional cloning of a cDNA encoding Mei2-like protein from Arabidopsis thaliana using a fission yeast pheromone receptor deficient mutant.

To isolate Arabidopsis cDNAs that encode signal transducers and components involved in the regulation of meiosis, a trans-complementation analysis was performed using a Schizosaccharomyces pombe meiosis-defective mutant in which the genes for pheromone receptors were disabled. One cDNA obtained in this screening encodes a polypeptide, named AML1, that shows significant similarity to S. pombe Mei2 protein and has three putative RNA-recognition motifs like as Mei2. Mei2 is involved in the regulation of meiosis in fission yeast. Northern blot analysis showed that the AML1 gene is expressed in each organ. The possible functions of AML1 are discussed.

[Efficacy and side effects of lidocaine by intravenous drip infusion in children with intractable seizures].

Intravenous drip infusions of lidocaine (IDIL; 1-5 mg/kg/h) were performed in ten patients with intractable seizures. The medication was very effective in five patients, whose seizures disappeared immediately after the treatment of IDIL. In four patients, the medication proved to be effective judging from decreased incidence of seizures. In one patient with intractable seizures, the therapy was not effective. In nine patients with the effective medication, five had generalized seizures and four had partial seizures. Side effects were observed in four patients. Two patients had muscle hypotonia, one had visual and auditory hallucination and another had bradycardia. These symptoms completely disappeared after the ceasing of IDIL. Serum concentrations of lidocaine at the appearance of the side effects ranged from 1.8 to 4.7 micrograms/ml, although the toxicity level is more than 5.0 micrograms/ml for arrhythmic adult patients. These findings suggest that the serum toxic level of lidocaine in children is different from that in adults, and that careful observation and appropriate management for the children with lidocaine therapy should be necessary, even though the serum level of lidocaine ranges within the therapeutic level.