Using microdensitometry to correlate cell morphology with the nuclear cycle in Ustilago maydis.

摘要

Nuclear staining and computer-assisted microdensitometry were used to investigate nuclear cycle events in U. maydis [U. zeae]. Vegetative cells were taken from liquid cultures at various points along the growth curve, attached to microscope slides,stained with 4',6-diamidino-2-phenylindole, and viewed using differential interference contrast and epifluorescence microscopy. Digital images of fluorescing nuclei were captured and inverted (negated) so that densitometry software could be used to determine the relative DNA content of the nuclei. When nuclei were grouped by relative density, 2 peaks, presumably corresponding to groups of nuclei before (1C) and after (2C) DNA synthesis, were observed. Correlating nuclear density with cell morphology showed that cells completed DNA synthesis before forming buds. The presence of some unbudded cells with the 1C amount of DNA suggested that there was a clearly defined G1 period. During early log phase, c. half of the cells in a culture were in G1. As the growth rate slowed, the percentage of cells in G1 increased sharply. This suggested that the length of time spent in G1 of varied with growth conditions. When cells from late log phase populations were subcultured into fresh medium, a lag period precededresumption of cell growth. Also, a significant percentage of these cells underwent a round of nuclear division and formed a central septum prior to bud formation. This resulted in a population of binucleate cells that budded asynchronously at both poles.