Fission yeast on the brink of meiosis.

The fission yeast Schizosaccharomyces pombe (S. pombe) is now well established as a versatile genetic model organism. It is widely used to analyse the basic eukaryotic cell cycle during vegetative growth and it is also well suited to studies on the elementary processes of sexual reproduction, including intercellular communication and signal transduction in zygote formation, as well as meiosis before sporulation. Systematic mutant screening has contributed much to our current understanding of unicellular differentiation in S. pombe, and structural analysis has revealed a simplified meiotic prophase with abundant crossing-over but no homologue synapsis. This article is a personal account of how this branch of fission yeast genetics has developed.

Stereotactic pallidotomy in a child with Hallervorden-Spatz disease. Case report.

The authors present a case of Hallervorden-Spatz disease (HSD) in a 10-year-old boy treated with stereotactic pallidotomy for control of severe dystonia. Hallervorden-Spatz disease is a rare type of neuraxonal dystrophy that can be familial or sporadic. This is the first case of HSD reported in the literature in which a pallidotomy was performed. The patient had progressively worsening dystonias and spasms that prevented useful function of his entire right side and eventually threatened his respiratory ability. Pre- and postoperative magnetic resonance images are presented along with electrophysiological recordings made in the globus pallidus at the time of surgery. Functional improvement in the use of the patient's limbs and relief from the painful dystonia were observed. Stereotactic pallidotomy should be considered as a potential treatment in the management of HSD.

FH3, a domain found in formins, targets the fission yeast formin Fus1 to the projection tip during conjugation.

Formins are involved in diverse aspects of morphogenesis, and share two regions of homology: FH1 and FH2. We describe a new formin homology region, FH3. FH3 is an amino-terminal domain that differs from the Rho binding site identified in Bni1p and p140mDia. The Schizosaccharomyces pombe formin Fus1 is required for conjugation, and is localized to the projection tip in cells of mating pairs. We replaced genomic fus1+ with green fluorescent protein (GFP)- tagged versions that lacked either the FH1, FH2, or FH3 domain. Deletion of any FH domain essentially abolished mating. FH3, but neither FH1 nor FH2, was required for Fus1 localization. An FH3 domain-GFP fusion protein localized to the projection tips of mating pairs. Thus, the FH3 domain alone can direct protein localization. The FH3 domains of both Fus1 and the S. pombe cytokinesis formin Cdc12 were able to localize GFP to the spindle pole body in half of the late G2 cells in a vegetatively growing population. Expression of both FH3-GFP fusions also affected cytokinesis. Overexpression of the spindle pole body component Sad1 altered the distribution of both Sad1 and the FH3-GFP domain. Together these data suggest that proteins at multiple sites can interact with FH3 domains.

F-actin distribution and function during sexual differentiation in Schizosaccharomyces pombe.

Sexual differentiation in Schizosaccharomyces pombe is induced from the G1 phase of the cell cycle by nitrogen starvation and the presence of mating pheromones. We describe the distribution of F-actin during sexual differentiation. Cortical F-actin dots have previously been shown to be restricted to one end of the rod shaped cell during the G1 phase of the cell cycle. Within half an hour of nitrogen starvation the distribution of cortical F-actin dots switched from being monopolar to bipolar. This was then reversed as the F-actin cytoskeleton repolarized so that cortical F-actin dots accumulated towards the projection tip at one end of the cell. Following cell fusion, F-actin dots were randomly scattered during the horsetail movement that precedes meiosis I and remained scattered until prometaphase or metaphase of meiosis II, when they concentrated around the nucleus. F-actin was seen on the lagging face of the nuclei which faced the partner nucleus during anaphase B of meiosis II. Early on in this anaphase F-actin was also seen on the opposite side of the nucleus, near the spindle pole body. F-actin accumulated within the spores in the mature ascus. Treatment with the actin depolymerising drug Latrunculin A showed that F-actin is required for cell fusion and spore formation. Latrunculin A treatment extended all stages from karyogamy to meiosis I. The S. pombe homologue of the actin binding protein profilin, Cdc3, was shown to be required for conjugation. Cdc3 co-localized with the formin related molecule Fus1 at the projection tip. The polarization of F-actin cortical dots to the projection tip was unaffected in the cdc3.124 mutant, but cdc3.124 mutant cells were unable to break down the cell walls between the two cells following agglutination.

Mutations in the fission yeast silencing factors clr4+ and rik1+ disrupt the localisation of the chromo domain protein Swi6p and impair centromere function.

Transcriptional silencing is known to occur at centromeres, telomeres and the mating type region in the nucleus of fission yeast, Schizosaccharomyces pombe. Mating-type silencing factors have previously been shown also to affect transcriptional repression within centromeres and to some extent at telomeres. Mutations in the clr4+, rik1+ and swi6+ genes dramatically reduce silencing at certain centromeric regions and cause elevated chromosome loss rates. Recently, Swi6p was found to co-localise with the three silent chromosomal regions. Here the involvement of clr4+, rik1+ and swi6+ in centromere function is investigated in further detail. Fluorescence in situ hybridisation (FISH) was used to show that, as in swi6 mutant cells, centromeres lag on late anaphase spindles in clr4 and rik1 mutant cells. This phenotype is consistent with a role for these three gene products in fission yeast centromere function. The Swi6 protein was found to be delocalised from all three silent chromosomal regions, and dispersed within the nucleus, in both clr4 and rik1 mutant cells. The phenotypic similarity observed in all three mutants is consistent with the products of both the clr4+ and rik1+ genes being required to recruit Swi6p to the centromere and other silent regions. Mutations in clr4, rik1 and swi6 also result in elevated sensitivity to reagents which destabilise microtubules and show a synergistic interaction with a mutation in the beta-tubulin gene (nda3). These observations suggest that clr4+ and rik1+ must play a role in the assembly of Swi6p into a transcriptionally silent, inaccessible chromatin structure at fission yeast centromeres which is required to facilitate interactions with spindle microtubules and to ensure normal chromosome segregation.

Two-step activation of meiosis by the mat1 locus in Schizosaccharomyces pombe.

The mat1 locus is a key regulator of both conjugation and meiosis in the fission yeast Schizosaccharomyces pombe. Two alternative DNA segments of this locus, mat1-P and mat1-M, specify the haploid cell types (Plus and Minus). Each segment includes two genes: mat1-P includes mat1-Pc and mat1-Pm, while mat1-M includes mat1-Mc and mat1-Mm. The mat1-Pc and mat1-Mc genes are responsible for establishing the pheromone communication system that mediates conjugation between P and M cells, while all four mat1 genes are required for meiosis in diploid P/M cells. Our understanding of the initiation of meiosis is based largely on indirect observations, and a more precise investigation of these events was required to define the interaction between the mat1 genes. Here we resolve this issue using synthetic pheromones and P/M strains with mutations in either mat1-Pc or mat1-Mc. Our results suggest a model in which the mat1 locus plays two roles in controlling meiosis. In the first instance, the mat1-Pc and mat1-Mc functions are required to produce the mating pheromones and receptors that allow the generation of a pheromone signal. This signal is required to induce the expression of mat1-Pm and mat1-Mm. This appears to be the major pheromone-dependent step in controlling meiosis since ectopic expression of these genes allows meiosis in the absence of mat1-Pc and mat1-Mc. The mat1-Pm and mat1-Mm products complete the initiation of meiosis by activating transcription of the mei3 gene.

Characterization of fus1 of Schizosaccharomyces pombe: a developmentally controlled function needed for conjugation.

In Schizosaccharomyces pombe, the fus1 mutation blocks conjugation at a point after cell contact and agglutination. The cell walls separating the mating partners are not degraded, which prevents cytoplasmic fusion. In order to investigate the molecular mechanism of conjugation, we cloned the fus1 gene and found that it is capable of encoding a 1,372-amino-acid protein with no significant similarities to other known proteins. Expression of the fus1 gene is regulated by the developmental state of the cells. Transcription is induced by nitrogen starvation and requires a pheromone signal in both P and M cell types. Consequently, mutants defective in the pheromone response pathway fail to induce fus1 expression. The ste11 gene, which encodes a transcription factor controlling expression of many genes involved in sexual differentiation, is also required for transcription of fus1. Furthermore, deletion of two potential Ste11 recognition sites in the fus1 promoter region abolished transcription, and expression could be restored when we inserted a different Ste11 site from the mat1-P promoter. Since this element was inverted relative to the fus1 element, we conclude that activation of transcription by Ste11 is independent of orientation. Although the fus1 mutant has a phenotype very similar to that of Saccharomyces cerevisiae fus1 mutants, the two proteins appear to have different roles in the process of cell fusion. Budding yeast Fus1 is a typical membrane protein and contains an SH3 domain. Fission yeast Fus1 has no features of a membrane protein, yet it appears to localize to the projection tip. A characteristic proline-rich potential SH3 binding site may mediate interaction with other proteins.

Posttraumatic childhood lumbosacral plexus neuropathy.

A 13-month-old male received crush injury to the abdomen resulting in paraparesis due to lumbosacral plexus neuropathy. The child was monitored with serial clinical examinations and electromyography/nerve conduction studies. He had complete clinical recovery. Lumbosacral plexus neuropathy is unusual in childhood and has not been previously reported as a result of abdominal trauma. This patient is presented with details of the clinical course, electrodiagnostic studies, discussion, and literature review.

Assessment of pheromone production and response in fission yeast by a halo test of induced sporulation.

We describe a rapid, sensitive and semi-quantitative plate assay for monitoring pheromone activity in the fission yeast Schizosaccharomyces pombe. It is based on the observation that meiosis requires stimulation by pheromone and exploits diploid strains that will only sporulate after addition of exogenous pheromone. The tester strains are heterozygous for mating type, are non-switching, and are mutated in one of the early subfunctions (either mat1-Mc or mat1-Pc), so that meiosis is only induced after exposure to exogenous pheromone (M-factor or P-factor, respectively). Pheromone activity is assessed as an iodine-positive halo of sporulation surrounding the pheromone source, and the width of the halo is related to the amount of pheromone being produced. The assay is sufficiently sensitive to monitor the low amount of M-factor produced by an M mam1 strain, and its sensitivity towards P-factor is greatly increased by using a hyper-sensitive tester strain lacking the Sxa2 protease that is believed to degrade this pheromone. We also demonstrate that the production of P-factor is very much stimulated by exposure of P cells to M-factor.

Mapping of additional markers in fission yeast, especially fus1 and three mfm genes.

The following genes of the fission yeast Schizosaccharomyces pombe have been mapped by tetrad analysis--chromosome arm I-L: mfm2, rad24, rad25; I-R: abc1, fus1, mfm1; II-L: mfm3; II-R: mam1, rad13. A hot-spot of meiotic recombination although not quite so active as suggested by previous maps, may be located between rad25 and aro5 on I-L.

Anterior fontanel herniation in group B streptococcus meningitis in newborns.

Brain herniation at any site is a rare complication of neonatal meningitis. Two cases of anterior fontanel herniation, associated with group B beta-hemolytic streptococcus meningitis are reported. This finding is a poor prognostic sign.

Reorientation of the distal region in linkage group IIR of fission yeast.

The genetic map of the fission yeast Schizosaccharomyces pombe has been revised in the distal region of chromosome arm IIR. The spo4 locus, hitherto considered the outermost marker, has been moved to an intermediate position. As a result, and in accordance with recent physical mapping data, the order of the entire distal subgroup of some 12 genetic markers is reversed relative to previously published gene maps.

The smt-0 mutation which abolishes mating-type switching in fission yeast is a deletion.

Mating-type switching in the fission yeast, S. pombe, is initiated by a DNA double-strand break (DSB) between the mat1 cassette and the H1 homology box. The mat1-cis-acting mutant, smt-0, abolishes mating-type switching and is shown here to be a 263-bp deletion. This deletion starts in the middle of the H1 homology box, 31 bp from the site of the DSB, and extends into the flanking region distal to mat1. The sequence of the region distal to H1 in the wild-type is also presented. In this region we observe a bias in the distribution of purine residues between the two DNA strands.

Functional conservation between Schizosaccharomyces pombe ste8 and Saccharomyces cerevisiae STE11 protein kinases in yeast signal transduction.

In fission yeast (Schizosaccharomyces pombe), the mat1-Pm gene, which is required for entry into meiosis, is expressed in response to a pheromone signal. Cells carrying a mutation in the ste8 gene are unable to induce transcription of mat1-Pm in response to pheromone, suggesting that the ste8 gene product functions in the signal transduction pathway. The ste8+ gene encodes a 659 amino acid putative protein kinase, which is identical to the previously identified byr2 suppressor of the ras1 defect. Furthermore, ste8+ is highly homologous to the Saccharomyces cerevisiae STE11 gene, which functions in signal transduction in budding yeast. Expression of the S. cerevisiae STE11 gene in S. pombe ste8 mutants restores the ability to transcribe mat1-Pm in response to pheromone. Also, such cells become capable of conjugation and sporulation. When mat1-Pm is artifically expressed from a heterologous promoter, ste8 mutant cells will enter meiosis. This demonstrates that the meiotic defect of ste8 mutants is due to the absence of the mat1-Pm gene product.

The ras1 function of Schizosaccharomyces pombe mediates pheromone-induced transcription.

Loss of ras1+ function renders fission yeast cells unable to undergo morphological changes in response to mating pheromones, whereas cells carrying activated mutations in ras1 are hyper-responsive. This has led to the suggestion that the ras1 gene product plays a role in mating pheromone signal transduction. Using partially purified M factor we demonstrate that the mat1-Pm gene, which controls entry into meiosis, is transcribed in response to a pheromone signal. Strains mutated in the ras1 gene or in ste6, the fission yeast homologue of Ras protein GDP/GTP exchange factor, are unable to induce transcription of mat1-Pm in response to M factor. Furthermore, an activated ras1val17 mutant exhibits a stronger induction of the mat1-Pm transcript. However, transcription still depends on nitrogen deprivation as well as on the presence of pheromone, showing that activation of the Ras1 protein alone does not substitute for any of these signals. The pat1-114 mutant bypasses the ras1/ste6 checkpoint, suggesting that activation of ras1 contributes to inactivation of the pat1 protein kinase.

Perioperative spinal canal narrowing in patients with Down's syndrome.

Two patients with Down's syndrome undergoing intracardiac operations had segmental and generalized myoclonic movements postoperatively and eventual brain death. Electroencephalography in 1 patient showed no seizure despite the presence of the myoclonic movements. Computed tomographic scan showed possible cerebellar hemorrhage. Ultrasound showed cerebral edema when the pupils became fixed and dilated. Because known postoperative neurologic complications could not fully explain the clinical course, and the myoclonic movements suggested spinal origin, we considered the possibility of atlantoaxial instability causing spinal cord damage related to perioperative head and neck positioning. Postmortem study on the second patient revealed 50% reduction of the spinal canal with hyperextension and 90-degree external rotation of the head and neck. In contrast, similar maneuvers in 3 infants without Down's syndrome resulted in only mild spinal canal narrowing. Although the myoclonic movements could be explained by spinal cord compression at the atlantoaxial level, the explanation for the eventual brain death is unclear. However, kinking of the vertebral arteries related to the positioning could have caused cerebellar ischemia, hemorrhage, and increased intracranial pressure. We believe that attention to the problem might bring further answers in the future.