Molecular mechanisms for regulation of microtubule architecture

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When centrosomes form mammalian cultured cells were add to Xenopus M phase egg extracts, Xenopus p48 (EF-1alpha) was found to accumulate to the centrosomes. This accumulation of p48 was not inhibited by anti-microtubule drugs, indicating that microtubules are not involved in the process of accumulation.We succeeded in cDNA cloning of Xenopus microtubule-associated protein p220 by using anti-p-220 monoclonal antibodies. The deduced amino acid sequence of p220 was similar to that of mammalian MAP4, suggesting that p220 is homologous to MAP4.We produced several monoclonal antibodies against Xenopus centrosomes. One of the antibodies, named W8-C3, recognized centrosomes alone in interphase cells, but reacted with spindle microtubules in addition to centrosomes in M phase cells. We isolated a few positive clones by screening Xenopus cDNA expression library with W8-C3. Sequencing part of one of the clones revealed that it is a Xenopus homolog of human PCM-1. Then, we isolated a full-length cDNA clone encoding Xenopus PCM-1 and determined its nucleotide sequence. We are now examining functions of Xenopus PCM-1 in regulation of microtubule dynamics and organization during cell cycle.