CELL CYCLE-DEPENDENT EXPRESSION OF THE CHO2 ANTIGEN, A MINUS-END DIRECTED KINESIN-LIKE MOTOR IN MAMMALIAN-CELLS

A 69 kDa protein present in the interphase centrosome and mitotic spin dle/pole was identified with the CHO2 monoclonal antibodies raised aga inst mitotic spindles isolated from Chinese hamster ovary (CHO) cells. Isolation and characterization of antigen-specific cDNAs and recombin ant proteins demonstrated that the protein is a minus-end-directed mic rotubule motor with the motor domain located at the C terminus. Affini ty-purified polyclonal antibodies prepared to bacterially expressed fu sion proteins revealed the presence of the antigen in interphase nucle i, and the degree of nude ar immunostaining intensity varied among cel ls at different cell cycle stages. In order to examine the change of a ntigen expression during the cell cycle, we prepared synchronized popu lations of CHO cells, double stained with CHO2 and PCNA (proliferating cell nuclear antigen) antibodies, and quantitated the amounts of nucl ear fluorescence using the MetaMorpho image analysis software package. Cells right after cell division contained nuclei with the lowest leve l of CHO2 immunofluorescence. The immunofluorescence intensity progres sively increases through G(1) to S, reaching a maximum level by the en d of G(2). The antibody uniformly stained the entire nuclear region, a nd the total amount of fluorescence detected in G(2) cells was greater than three rimes that of G(1) cells. Cell cycle dependent accumulatio n of the CHO2 antigen was further confirmed by immunoblot analysis of the protein included in whole cell extracts and nuclei isolated from s ynchronized CHO cells. Northern blot analysis showed that, although th e CHO2-transcript accumulated during later stages of the cell cycle, i ts abundance declined through G(1) to S, and was lowest in cells at th e early S phase. The difference in the expression pattern of the antig en protein and its transcript may suggest the presence of multiple mec hanisms controlling the level of CHO2 antigen during the course of the cell cycle.