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Nucleic Acids Research, 2001, Vol. 29, No. 18 3796-3803
© 2001 Oxford University Press

Creation and characterization of temperature-sensitive CENP-C mutants in vertebrate cells

Tatsuo Fukagawa*, Vinciane Regnier1 and Toshimichi Ikemura

National Institute of Genetics and Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan and 1Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK

CENP-C is an evolutionarily conserved centromere protein that is thought to be an important component in kinetochore assembly in vertebrate cells. However, the functional role of CENP-C in cell cycle progression remains unclear. To further understand CENP-C function, we developed a method incorporating the hyper-recombinogenic chicken B lymphocyte cell line DT40 to create several temperature-sensitive CENP-C mutants in DT40 cells. We found that, under restrictive conditions, one temperature-sensitive mutant, ts4-11, displayed metaphase delay and chromosome missegregation but proceeded through the cell cycle until arrest at G1 phase. Furthermore, ts4-11 cells were transfected with a human HeLa cell cDNA library maintained in a retroviral vector, and genes that suppressed the temperature-sensitive phenotype were identified. One of these suppressor genes encodes SUMO-1, which is a ubiquitin-like protein. This finding suggests that SUMO-1 may be involved in centromere function in vertebrate cells. The novel strategy reported here will be useful and applicable to a wide range of proteins that have general cell-autonomous function in vertebrate cells.

* To whom correspondence should be addressed. Tel: +81 559 81 6792; Fax: +81 559 81 6794; Email: tfukagaw{at}lab.nig.ac.jp


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