A temperature-sensitive mutation of the Schizosaccharomyces pombe gene nuc2+ that encodes a nuclear scaffold-like protein blocks spindle elongation in mitotic anaphase.
A temperature-sensitive mutant nuc2-663 of the fission yeast Schizosaccharomyces pombe specifically blocks mitotic spindle elongation at restrictive temperature so that nuclei in arrested cells contain a short uniform spindle (approximately 3-micron long), which runs through a metaphase plate-like structure consisting of three condensed chromosomes. In the wild-type or in the mutant cells at permissive temperature, the spindle is fully extended approximately 15-micron long in anaphase. The nuc2' gene was cloned in a 2.4-kb genomic DNA fragment by transformation, and its complete nucleotide sequence was determined. Its coding region predicts a 665-residues internally repeating protein (76.250 mol wt). By immunoblots using anti-sera raised against lacZ-nuc2+ fused proteins, a polypeptide (designated p67; 67,000 mol wt) encoded by nuc2+ is detected in the wild-type S. pombe extracts; the amount of p67 is greatly increased when multi-copy or high-expression plasmids carrying the nuc2+ gene are introduced into the S. pombe cells. Cellular fractionation and Percoll gradient centrifugation combined with immunoblotting show that p67 cofractionates with nuclei and is enriched in resistant structure that is insoluble in 2 M NaCl, 25 mM lithium 3,5'-diiodosalicylate, and 1% Triton but is soluble in 8 M urea. In nuc2 mutant cells, however, soluble p76, perhaps an unprocessed precursor, accumulates in addition to insoluble p67. The role of nuc2+ gene may be to interconnect nuclear and cytoskeletal functions in chromosome separation.
Hirano
T
T
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Hiraoka
Y
Y
Yanagida
M
M
D000595:Amino Acid Sequence; D000705:Anaphase; D064191:Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome; D001483:Base Sequence; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000235:genetics* / Q000502:physiology; D005800:Genes, Fungal; D007118:Immunoassay; D008938:Mitosis; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics* / Q000502:physiology; D010641:Phenotype; D010957:Plasmids; D004718:Saccharomycetales / Q000235:genetics*; D012568:Schizosaccharomyces / Q000166:cytology / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins; D008941:Spindle Apparatus / Q000502:physiology*; D013696:Temperature; D014170:Transformation, Genetic
D064191:Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome; D005656:Fungal Proteins; C577354:NUC2 protein, S pombe; D009687:Nuclear Proteins; D029702:Schizosaccharomyces pombe Proteins