Creation and characterization of temperature-sensitive CENP-C mutants in vertebrate cells.
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 G(1) 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.
Fukagawa
T
T
National Institute of Genetics and Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan. tfukagaw@lab.nig.ac.jp
Regnier
V
V
Ikemura
T
T
D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000235:genetics / Q000502:physiology; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D002503:Centromere / Q000378:metabolism; D002645:Chickens; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000502:physiology*; D006801:Humans; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D025842:SUMO-1 Protein; D013489:Suppression, Genetic; D013696:Temperature; D013997:Time Factors; D014162:Transfection; D014452:Ubiquitins / Q000235:genetics / Q000502:physiology
D002868:Chromosomal Proteins, Non-Histone; D025842:SUMO-1 Protein; D014452:Ubiquitins; C060905:centromere protein C