RNAi and Ino80 complex control rate limiting translocation step that moves rDNA to eroding telomeres.

The discovery of HAATIrDNA, a telomerase-negative survival mode in which canonical telomeres are replaced with ribosomal DNA (rDNA) repeats that acquire chromosome end-protection capability, raised crucial questions as to how rDNA tracts 'jump' to eroding chromosome ends. Here, we show that HAATIrDNA formation is initiated and limited by a single translocation that juxtaposes rDNA from Chromosome (Chr) III onto subtelomeric elements (STE) on Chr I or II; this rare reaction requires RNAi and the Ino80 nucleosome remodeling complex (Ino80C), thus defining an unforeseen relationship between these two machineries. The unique STE-rDNA junction created by this initial translocation is efficiently copied to the remaining STE chromosome ends, independently of RNAi or Ino80C.

Generation of temperature sensitive mutations with error-prone PCR in a gene encoding a component of the spindle pole body in fission yeast.

Temperature-sensitive (ts) mutants provide powerful tools for investigation of cellular functions of essential genes. We report here asimple procedure to generate ts mutations using error-prone PCR within that encodes aspindle pole body (SPB) component in . This manipulation is not restricted to , and can be suited to any essential genes involved in other processes.

Distinct 'safe zones' at the nuclear envelope ensure robust replication of heterochromatic chromosome regions.

Chromosome replication and transcription occur within a complex nuclear milieu whose functional subdomains are beginning to be mapped out. Here we delineate distinct domains of the fission yeast nuclear envelope (NE), focusing on regions enriched for the inner NE protein, Bqt4, or the lamin interacting domain protein, Lem2. Bqt4 is relatively mobile around the NE and acts in two capacities.

RNAi drives nonreciprocal translocations at eroding chromosome ends to establish telomere-free linear chromosomes.

The identification of telomerase-negative HAATI (heterochromatin amplification-mediated and telomerase-independent) cells, in which telomeres are superseded by nontelomeric heterochromatin tracts, challenged the idea that canonical telomeres are essential for chromosome linearity and raised crucial questions as to how such tracts translocate to eroding chromosome ends and confer end protection. Here we show that HAATI arises when telomere loss triggers a newly recognized illegitimate translocation pathway that requires RNAi factors. While RNAi is necessary for the translocation events that mobilize ribosomal DNA (rDNA) tracts to all chromosome ends (forming "HAATI" chromosomes), it is dispensable for HAATI maintenance.

Synergistic role of fission yeast Alp16GCP6 and Mzt1MOZART1 in γ-tubulin complex recruitment to mitotic spindle pole bodies and spindle assembly.

In fission yeast, γ-tubulin ring complex (γTuRC)-specific components Gfh1(GCP4), Mod21(GCP5), and Alp16(GCP6) are nonessential for cell growth. Of these deletion mutants, only alp16Δ shows synthetic lethality with temperature-sensitive mutants of Mzt1(MOZART1), a component of the γTuRC required for recruitment of the complex to microtubule-organizing centers. γ-Tubulin small complex levels at mitotic spindle pole bodies (SPBs, the centrosome equivalent in fungi) and microtubule levels for preanaphase spindles are significantly reduced in alp16Δ cells but not in gfh1Δ or mod21Δ cells.

Fission yeast nucleolar protein Dnt1 regulates G2/M transition and cytokinesis by downregulating Wee1 kinase.

Cytokinesis involves temporally and spatially coordinated action of the cell cycle, cytoskeletal and membrane systems to achieve separation of daughter cells. The septation initiation network (SIN) and mitotic exit network (MEN) signaling pathways regulate cytokinesis and mitotic exit in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Previously, we have shown that in fission yeast, the nucleolar protein Dnt1 negatively regulates the SIN pathway in a manner that is independent of the Cdc14-family phosphatase Clp1/Flp1, but how Dnt1 modulates this pathway has remained elusive.

Fission yeast MOZART1/Mzt1 is an essential γ-tubulin complex component required for complex recruitment to the microtubule organizing center, but not its assembly.

γ-Tubulin plays a universal role in microtubule nucleation from microtubule organizing centers (MTOCs) such as the animal centrosome and fungal spindle pole body (SPB). γ-Tubulin functions as a multiprotein complex called the γ-tubulin complex (γ-TuC), consisting of GCP1-6 (GCP1 is γ-tubulin). In fungi and flies, it has been shown that GCP1-3 are core components, as they are indispensable for γ-TuC complex assembly and cell division, whereas the other three GCPs are not.

Nonself recognition through intermolecular disulfide bond formation of ribonucleotide reductase in neurospora.

Type I ribonucleotide reductases (RNRs) are conserved across diverse taxa and are essential for the conversion of RNA into DNA precursors. In Neurospora crassa, the large subunit of RNR (UN-24) is unusual in that it also has a nonself recognition function, whereby coexpression of Oak Ridge (OR) and Panama (PA) alleles of un-24 in the same cell leads to growth inhibition and cell death. We show that coexpressing these incompatible alleles of un-24 in N.

Spatiotemporal regulations of Wee1 at the G2/M transition.

Wee1 is a protein kinase that negatively regulates mitotic entry in G2 phase by suppressing cyclin B-Cdc2 activity, but its spatiotemporal regulations remain to be elucidated. We observe the dynamic behavior of Wee1 in Schizosaccharomyces pombe cells and manipulate its localization and kinase activity to study its function. At late G2, nuclear Wee1 efficiently suppresses cyclin B-Cdc2 around the spindle pole body (SPB).

Localization of gene products using a chromosomally tagged GFP-fusion library in the fission yeast Schizosaccharomyces pombe.

We constructed a library of chromosomally-tagged green fluorescent protein (GFP) fusions in the fission yeast Schizosaccharomyces pombe. This library contains 1058 strains. In each strain, the coding sequence of GFP is integrated at the 3'-end of a particular chromosomal ORF such that the full-length GFP fusion construct is expressed under the control of the original promoter.

Fission yeast kinesin-8 Klp5 and Klp6 are interdependent for mitotic nuclear retention and required for proper microtubule dynamics.

Fission yeast has two kinesin-8s, Klp5 and Klp6, which associate to form a heterocomplex. Here, we show that Klp5 and Klp6 are mutually dependent on each other for nuclear mitotic localization. During interphase, they are exported to the cytoplasm.

Fission yeast dam1-A8 mutant is resistant to and rescued by an anti-microtubule agent.

The Dam1/DASH outer kinetochore complex is required for high-fidelity chromosome segregation in budding and fission yeast. Unlike budding yeast, the fission yeast complex is non-essential, however it promotes bipolar microtubule attachment in conjunction with microtubule-depolymerising kinesin-8 Klp5 and Klp6. Here, we screened for dam1 temperature sensitive mutants in a klp5 null background and identified dam1-A8 that contains two amino acid substitutions in the C-terminus (H126R and E149G).

Nuclear localization of barrier-to-autointegration factor is correlated with progression of S phase in human cells.

Barrier-to-autointegration factor (BAF) is a conserved metazoan protein that plays a critical role in retrovirus infection. To elucidate its role in uninfected cells, we first examined the localization of BAF in both mortal and immortal or cancerous human cell lines. In mortal cell lines (e.

The carboxy-terminus of Alp4 alters microtubule dynamics to induce oscillatory nuclear movement led by the spindle pole body in Schizosaccharomyces pombe.

Alp4 is an essential component of the S. pombe gamma-tubulin complex. Overproduction of the carboxy-terminus of Alp4 induces oscillatory nuclear movement led by the spindle pole body (SPB).

Modulation of Alp4 function in Schizosaccharomyces pombe induces novel phenotypes that imply distinct functions for nuclear and cytoplasmic gamma-tubulin complexes.

The gamma-tubulin complex acts as a nucleation unit for microtubule assembly. It remains unknown, however, how spatial and temporal regulation of the complex activity affects microtubule-mediated cellular processes. Alp4 is one of the essential components of the S.

POLYTENE CHROMOSOMES ISOLATED FROM NUCLEI OF TOKUNAGAYUSURIKA AKAMUSHI (DIPTERA, CHIRONOMIDAE): STRUCTURAL TRANSFORMATION CAUSED BY SALT, DETERGENT POLYANIONS AND ENZYMES.

Polytene chromosomes of Tokunagayusurika akamushi (Chironomidae) were isolated from nuclei of the salivary glands. The isolated chromosomes retained their normal morphology, revealing bands by phase contrast microscopy. Extensive structural transformations were observed when the isolated chromosomes were immersed in solutions of high salt concentration, polyanions (dextran sulfate and heparin), the ionic detergent SDS (0.