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First published online 3 February 2004
doi: 10.1242/jcs.00946

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A novel RING-finger-like protein Ini1 is essential for cell cycle progression in fission yeast

Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, PO Box 016129, Miami, Florida 33101-1019, USA

View larger version (32K): [in a new window]   Fig. 1. Identification and characterization of the ini1+ gene in S. pombe. (A) Structure of the fission yeast ini1+ gene encoded by the antisense strand of chromosome I located between positions 3190 and 2686 of cosmid 23H3. E1-E4 corresponds to exons 1 through 4; the number of amino acids encoded in each exon is indicated. I1-I3 refers to introns 1 through 3; the number of base pairs in each intron is indicated. (B) Alignment of Ini amino acid sequences from human, budding yeast and fission yeast using the ClustalW interactive Multiple Sequence Alignment Program from EBI (European Bioinformatics Institute, http://www.ebi.ac.uk). Identical residues are boxed in black. Conserved residues are boxed in gray.

View larger version (37K): [in a new window]   Fig. 2. Germination of spores disrupted for the ini1+ gene. (A) Phase contrast images of spores derived from an ini1+/ini1 tetrad upon germination in YE medium. A 2:2 segregation pattern is observed. (B) DNA content of germinating haploid spores derived from ini1+/ini1 diploids grown in either the presence of uracil, to allow all spores to germinate (left), or in the absence of uracil, to restrict germination to only those spores deleted for ini1+ (right). The positions of 1C and 2C DNA content are indicated. Cells were collected at 2-hour intervals for 12 hours and processed for flow cytometry (FACS) analysis as previously described (Sazer, 1990).

View larger version (21K): [in a new window]   Fig. 3. Cells depleted of ini1+ arrest in the G2/M phase of the cell cycle. (A) DAPI-stained ini1 cells containing the wild-type ini1+ gene under the control of the thiamine repressible promoter Rep81 (ini1), grown in the absence (left panel) or presence (right panel) of thiamine. (B) Cell number increase for ini1 in the presence or absence of thiamine. (C) DNA content of the cells described in A, 12 hours after addition of thiamine.

View larger version (73K): [in a new window]   Fig. 4. Sub-cellular localization of Ini1. GFP-tagged Ini1 was localized predominantly to the nucleus of ini1 cells rescued by pRep3xGFP-Ini1 as detected by fluorescence microscopy. (A) DAPI staining; (B) GFP. Arrowheads indicate position of nucleolus.

View larger version (39K): [in a new window]   Fig. 5. Cell cycle arrest in the absence of Ini1 is Wee1 dependent. (A) DAPI images of (a) wild-type cells, (b) wee1-50ini1 cells, grown in the absence of thiamine, and (c) ini1 and (d) wee1-50ini1 cells, grown in the presence of thiamine for 12 hours. All cells were shifted to 36°C at the time of thiamine addition to c and d. Arrowheads in d indicate cells that have undergone an aberrant mitosis. Scale bar: 5 µm. (B) Quantitation of the results in A. Cell length was measured for >100 cells from two independent experiments 12 hours after shifting to 36°C in the absence (a,b) or presence (c,d) of thiamine. Data presented as mean ± s.e.m. as indicated by error bars. (C) Viability of ini1 and wee1-50ini1 following repression of ini1+ gene expression. All strains grown at 36°C were treated with thiamine for the indicated times. Open circles, ini1–th cells in the absence of thiamine; open squares, wee1-50ini1 in the absence of thiamine; closed squares, ini1 cells in the presence of thiamine; closed circles, wee1-50ini1 in the presence of thiamine. Data presented as mean±s.e.m. as indicated by error bars.

View larger version (60K): [in a new window]   Fig. 6. The ini1+ gene is required for pre-mRNA splicing. (A-F) RT-PCR analysis of cwf8+, cdc2+ and tfIId+, ade2+, nda3+ and rpl7+ RNAs isolated from the temperature sensitive prp2 mutant cells at the permissive (25°C) or the restrictive (36°C) temperature, and from Rep81ini1+ cells grown in the absence (–Th) or presence (+Th) of thiamine for 12 hours. Unspliced and processed mRNAs are indicated. DNA molecular mass markers (M) are also shown. (G) Northern blot analysis of pre-mRNA and mRNA for the gene encoding U6 snRNA isolated from prp2-1 cells at 25°C and 36°C and from ini1 cells with or without thiamine for 12 hours. See Materials and Methods for details. Lower panel shows ethidium bromide-stained gel of 5S RNA, as a loading control.

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