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. How ever, the functional role of CENP-C in cell cycle progression remains uncle ar. To further understand CENP-C function, we developed a method incorporat ing 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, dis played metaphase delay and chromosome missegregation but proceeded through the cell cycle until arrest at G, phase. Furthermore, ts4-11 cells were tra nsfected with a human HeLa cell cDNA library maintained in a retroviral vec tor, and genes that suppressed the temperature-sensitive phenotype were ide ntified. One of these suppressor genes encodes SUMO-1, which is a ubiquitin -like protein. This finding suggests that SUMO-1 may be involved in centrom ere function in vertebrate cells. The novel strategy reported here will be useful and applicable to a wide range of proteins that have general cell-au tonomous function in vertebrate cells.