Properties of the interphase microtubule organizing centers (MTOCs) of CHO cells were investigated using indirect immunofluorescence staining technique.Microtubule proteins were isolated from swine brain by three cyc...Properties of the interphase microtubule organizing centers (MTOCs) of CHO cells were investigated using indirect immunofluorescence staining technique.Microtubule proteins were isolated from swine brain by three cycles of assembly-disassembly- 6S tubulin was purified by chromatograph y through cellulose phosphate p-11 column. Rabbits were immunized with the purified 6S tubulin and the anti-6S tubulin antiserum thus prepared was used in the indirect immunofluorescence staining of nuclei With double thymidine block method, we obtained cells at G1/S, S and G2 phases of cell cycle and isolated nuclei from cells of each pltase, Immunofluorescence staining of MTOCs could hardly he detected in the Triton X-100 treated cells nor in nuclei of any phase after preincubation with purified 6S tubulin alone. Yet,if the Triton X-100 treated cells or nuclei were incubated, in the presence of 5 mM ATP, with maturation promoting factor (MPF)isolated from mature oocytes of Xenopus for 40 minutes before the addition of the 6S tubulin, specifically stained MTOCs were clearly demonstrated in S and G2 nuclei Furthermore, microbubules could be observed radiating from the MTOCs in late S and G2 nuclei. However, alkaline pHospHatase treatment of the nuclei which had been incubated with the activated MPF prohibited the staining of MTOCs, and MPF isolated from premature oocytes failed to contribute to the immunostaining of MTOCs. Our results thus suggest that the transformation of kinetochores into MTOCs during cell cycle requires activated MPF and phosphorylation of proteins.展开更多
文摘Properties of the interphase microtubule organizing centers (MTOCs) of CHO cells were investigated using indirect immunofluorescence staining technique.Microtubule proteins were isolated from swine brain by three cycles of assembly-disassembly- 6S tubulin was purified by chromatograph y through cellulose phosphate p-11 column. Rabbits were immunized with the purified 6S tubulin and the anti-6S tubulin antiserum thus prepared was used in the indirect immunofluorescence staining of nuclei With double thymidine block method, we obtained cells at G1/S, S and G2 phases of cell cycle and isolated nuclei from cells of each pltase, Immunofluorescence staining of MTOCs could hardly he detected in the Triton X-100 treated cells nor in nuclei of any phase after preincubation with purified 6S tubulin alone. Yet,if the Triton X-100 treated cells or nuclei were incubated, in the presence of 5 mM ATP, with maturation promoting factor (MPF)isolated from mature oocytes of Xenopus for 40 minutes before the addition of the 6S tubulin, specifically stained MTOCs were clearly demonstrated in S and G2 nuclei Furthermore, microbubules could be observed radiating from the MTOCs in late S and G2 nuclei. However, alkaline pHospHatase treatment of the nuclei which had been incubated with the activated MPF prohibited the staining of MTOCs, and MPF isolated from premature oocytes failed to contribute to the immunostaining of MTOCs. Our results thus suggest that the transformation of kinetochores into MTOCs during cell cycle requires activated MPF and phosphorylation of proteins.
