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Optimization of culture and treatment conditions for reproducible synchronization of the cell cycle in maize cells with hydroxyurea is described. Flow cytometric measurements of relative DNA contents revealed that, following the hydroxyurea block, around 55% of the cells were in the G1 phase (2C), 30% in the S phase (2C 4C) and 15% in the G2 phase (4C) of the cell cycle. The highest frequencies of cells in the S phase could be observed 2 h after removal of hydroxyurea (35-40%). The cells reached increased G2 phase frequencies (60-70%) between 8-14 h, while the maximum number of mitotic cells (12-14%) were found between 14-17 h. Northern analysis of total RNA from the synchronized cells indicated an increased level of transcripts from two histone (H3Zm, H4Zm) genes during the S-phase. The changes in the mRNA levels of the maize cyclin variants CycB1;zm;1, CycB1;zm;2 and CycA1;zm;1 support the classification of these cyclins as mitotic cyclins with transcript accumulation during the G 2 /M cell cycle phases. Using the maize retinoblastoma (ZmRb-1) cDNA as hybridization probe, two transcripts were detected with different hybridization intensity: the smaller, more abundant transcript was recognized during the whole cell cycle with an increase after the release of the cells from the hydroxyurea block, while the larger mRNA could only be detected for 8h after removal of hydroxyurea. The ZmRb-1 gene might respond to the increase of the frequency of cells in the S phase or to the re-addition of conditioned medium to the cells after hydroxyurea removal.

journal of experimental botany

Partial synchronization of cell division in cultured maize (Zea mays L.) cells: differential cyclin, cdc2, histone, and retinoblastoma transcript accumulation during the cell cycle