Abstract. The fission yeast Schizosaccharomyces pombe divides by medial fission and, like many higher eukaryotic cells, requires the function of an F-actin contractile ring for cytokinesis. In S. pombe, a class of cdc-mutants defective for cytokinesis, but not for DNA replication, mitosis, or septum synthesis, have been identified. In this paper, we present the characterization of one of these mutants, cdc3-124. Temperature shift experiments reveal that mutants in cdc3 are incapable of forming an F-actin contractile ring. We have molecularly cloned cdc3 and used the cdc3 + genomic DNA to create a strain carrying a cdc3 null mutation by homologous recombination in vivo. Cells bearing a cdc3-null allele are inviable. They arrest the cell cycle at cytokinesis without forming a contractile ring, DNA sequence analysis of the cdc3 + gene reveals that it encodes profilin, an actin-monomer-binding protein. In light of recent studies with profilins, we propose that Cdc3-profilin plays an essential role in cytokinesis by catalyzing the formation of the F-actin contractile ring. Consistent with this proposal are our observations that Cdc3-profilin localizes to the medial region of the cell where the F-actin contractile ring forms, and that it is essential for F-actin ring formation. Cells overproducing Cdc3-profilin become elongated, dumbbell shaped, and arrest at cytokinesis without any detectable F-actin staining. This effect of Cdc3-profilin overproduction is relieved by introduction of a multicopy plasmid carrying the actin encoding gene, act1 +. We attribute these effects to potential sequesteration of actin monomers by profilin, when present in excess.T I~E three major landmark events in the eukaryotic cell cycle are (a) replication of the genetic material that occurs in S phase; (b) partitioning of the replicated DNA to the daughter nuclei during M phase; and (c) division of the cell itself to produce two daughter cells as a result of cytokinesis. The last 10 yr have seen tremendous advances in our understanding of the molecular nature of processes occurring during the S and M phases and the controis that regulate entry into S and M phases. By contrast, the mechanisms and regulations that govern the third major landmark event, cytokinesis, remain only poorly understood.Much of the progress in our understanding of the controls that regulate S and M phases stem from genetic studies in the fission yeast Schizosaccharomyces pombe. Several lines of evidence suggest that this yeast should also be an ideal system with which to study cytokinesis. First, S. pombe cells divide by medial fission (Nurse, 1985) and, like many higher eukaryotic cells, produce equally sized daughter cells after cytokinesis. Second, cytokinesis in fission yeast, akin to

The Schizosaccharomyces pombe cdc5+ gene was identified in the first screen for cell division cycle mutants in this yeast. The cdc5+ gene was reported to be required for nuclear division but because of its modest elongation and leaky nature at the non‐permissive temperature, it was not investigated further. Here, we report the characterization of the single allele of this gene, cdc5‐120, in more detail. The mutant arrests with a 2N DNA content and a single interphase nucleus. Further genetic analyses suggest that cdc5+ gene function is essential in the G2 phase of the cell cycle. We have cloned and sequenced the cdc5+ gene. The deduced protein sequence predicts that Cdc5 is an 87 kDa protein and contains a region sharing significant homology with the DNA binding domain of the Myb family of transcription factors. Deletion mapping of the cdc5+ gene has shown that the N‐terminal 232 amino acids of the protein, which contain the Myb‐related region, are sufficient to complement the cdc5ts strain. A cdc5 null mutant was generated by homologous recombination. Haploid cells lacking cdc5+ are inviable, indicating that cdc5+ is an essential gene. A fusion protein consisting of bacterial glutathione S‐transferase joined in‐frame to the N‐terminal 127 amino acids of the Cdc5 protein is able to bind to DNA cellulose at low salt concentrations. This evidence suggests that cdc5+ might encode a transcription factor whose activity is required for cell cycle progression and growth during G2.

Optimal lysosome function requires maintenance of an acidic pH maintained by proton pumps in combination with a counterion transporter such as the Cl À /H þ exchanger, CLCN7 (ClC-7), encoded by CLCN7. The role of ClC-7 in maintaining lysosomal pH has been controversial. In this paper, we performed clinical and genetic evaluations of two children of different ethnicities. Both children had delayed myelination and development, organomegaly, and hypopigmentation, but neither had osteopetrosis. Whole-exome and-genome sequencing revealed a de novo c.2144A>G variant in CLCN7 in both affected children. This p.Tyr715Cys variant, located in the C-terminal domain of ClC-7, resulted in increased outward currents when it was heterologously expressed in Xenopus oocytes. Fibroblasts from probands displayed a lysosomal pH approximately 0.2 units lower than that of control cells, and treatment with chloroquine normalized the pH. Primary fibroblasts from both probands also exhibited markedly enlarged intracellular vacuoles; this finding was recapitulated by the overexpression of human p.Tyr715Cys CLCN7 in control fibroblasts, reflecting the dominant, gain-of-function nature of the variant. A mouse harboring the knock-in Clcn7 variant exhibited hypopigmentation, hepatomegaly resulting from abnormal storage, and enlarged vacuoles in cultured fibroblasts. Our results show that p.Tyr715Cys is a gain-of-function CLCN7 variant associated with developmental delay, organomegaly, and hypopigmentation resulting from lysosomal hyperacidity, abnormal storage, and enlarged intracellular vacuoles. Our data supports the hypothesis that the ClC-7 antiporter plays a critical role in maintaining lysosomal pH.

Dendritic cells (DCs) are powerful antigen-presenting cells that process antigens and present peptide epitopes in the context of the major histocompatibility complex molecules to generate immune responses. DCs are being studied as potential anticancer vaccines because of their ability to present antigens to naïve T cells and to stimulate the expansion of antigenspecific T-cell populations. We investigated an antitumor vaccination using DCs modified by transfer of a nonsignaling neu oncogene, a homologue of human HER-2/neu, in a transgenic model of breast cancer. BALB-neuT mice develop breast cancers as a consequence of mammary gland-specific expression of an activated neu oncogene. We vaccinated BALB-neuT mice with bone marrow-derived DCs transduced with Ad.Neu, a recombinant adenovirus expressing a truncated neu oncoprotein. The vaccine stimulated the production of specific anti-neu antibodies, enhanced interferon-␥ expression by T cells, and prevented or delayed the onset of mammary carcinomas in the mice. Over 65% of vaccinated mice remained tumor free at 28 weeks of age, whereas all of the mice in the control groups developed tumors. When challenged with a neu-expressing breast cancer cell line, vaccinated tumor-free animals had delayed tumor growth compared with controls. The antitumor effect of the vaccine was specific for expression of neu. Studies showed that CD4؉ T cells were required in order to generate antitumor immunity. Importantly, the effectiveness of the vaccine was not diminished by preexisting immunity to adenovirus, whereas the protection afforded by vaccination that used direct injection of Ad.Neu was markedly reduced in mice with antiadenovirus antibody titers. DCs modified by recombinant adenoviruses expressing tumor-associated antigens may provide an effective antitumor vaccination strategy.

A DNA fragment which carries the his3 gene of Schizosaccharomyces pombe has been isolated and characterized for use as a selectable marker in transformations. The his3 gene encodes the imidazole acetol phosphate transaminase enzyme (E.C.2.6.1.9), which is responsible for converting imidazole acetol-P to histidinol-P in step 8 of histidine biosynthesis. The nucleotide sequences of a 2196 bp gene fragment and a corresponding cDNA clone were determined. Three intron sequences punctuate the 1451 bp coding region which generates a predicted polypeptide of 384 amino acids with a molecular mass of 42736 daltons. Northern analysis of his3 mRNAs indicates that the transcript is approximately 1.6 kb in size. Steady-state levels are down-regulated by nitrogen limitation but are unaffected by histidine starvation. The deduced amino acid sequence was compared to the Saccharomyces cerevisiae HIS5, Escherichia coli HisC, and Salmonella typhimurium HisC proteins, all of which are imidazole acetol phosphate transaminases. The S. pombe his3 protein was 49.5% identical to the S. cerevisiae HIS5 protein and 21.5% identity was found when all four proteins were compared. The shuttle vector pBG1 was constructed by subcloning the smallest functional region of his3 and the S. pombe ars1 sequence into pUC18 for use in transformation of His3--S. pombe strains. New S. pombe strains in which the his3 gene was deleted have also been constructed.