USING MICRODENSITOMETRY TO CORRELATE CELL MORPHOLOGY WITH THE NUCLEAR-CYCLE IN USTILAGO-MAYDIS

Nuclear cycle events are usually studied using H-3-thymidine, which ce lls take up from culture medium and incorporate into DNA. Because fung i do not incorporate exogenously supplied thymidine, other methods mus t be used for these organisms. The experiments described here utilized nuclear staining and computer-assisted microdensitometry. Vegetative Ustilago maydis cells were taken from liquid cultures at various point s along the growth curve, attached to microscope slides, stained with 4',6-diamidino-2-phenylindole, and viewed using Differential Interfere nce Contrast and epifluorescence microscopy. Digital images of fluores cing nuclei were captured and inverted (negated) so that densitometry software could be used to determine the relative DNA content of the nu clei. When nuclei were grouped by relative density, two peaks presumab ly corresponding to groups of nuclei before (1C) and after (2C) DNA sy nthesis were observed. Correlating nuclear density with cell morpholog y showed that cells complete DNA synthesis before beginning to form bu ds. The presence of a number of unbudded cells with the 1C amount of D NA indicated that there was a clearly defined G1 period. During early log phase, approximately half of the cells in a culture were in G1. As the growth rate slowed, the percentage of cells in G1 increased drama tically. This indicates that the length of time spent in G1 varies wit h growth conditions in U. maydis. When cells from late log phase popul ations were subcultured into fresh medium, a lag period preceded resum ption of cell growth. In addition, a significant percentage of these c ells underwent a round of nuclear division and formed a central septum prior to bud formation. This resulted in a population of binucleate c ells that budded asynchronously at both poles.