Cycloheximide was used to study various aspects of cellular metabolism in root meristem cells of a single-cross hybrid, Seneca-60. Changes in the mitotic index (M.I.) were monitored for up to 8 hours at 30-minute intervals under three different conditions which varied in time or concentration of cycloheximide treatment. In all cases, a peak in mitotic activity occurred early in the treatment which was apparently due to an increase in the number of cells at metaphase. Root tips subjected to 7.5 µg/ml of cycloheximide for either 60 or 120 minutes demonstrated this peak at 60 minutes. Following the peak, the M.I. dropped rapidly to a value well below the control before recovering to the control value. The time of recovery was much longer for the 120-minute than for the 60-minute treatment. Using a cycloheximide concentration of 75.0 µg/ml for 60 minutes, the M.I. peak occurred at 15 minutes followed by a rapid drop and eventual recovery similar to the 120-minute treatment at the lower cycloheximide concentration.
The very low frequency of anaphases and high frequency of metaphases relative to the control (Table 1) at the peak of mitotic activity was attributed to a block in metaphase which either prevented or decreased the number of cells leaving metaphase. The recovery of anaphases following removal of the chemical further supports this suggestion. The drop in mitotic activity after 60 minutes in the cycloheximide-treated root tips is attributed to a transition point in G2 since the decrease in M.I. was due to a decline in the number of cells entering prophase. The duration of treatment, the length of time before the prophase frequency began to decrease, and the length of time until it reached the minimum value are all very similar between treatments (Table 1) and suggest that in maize, at least one cycloheximide-inducible, G2 transition point is about 1-1.5 hours before the onset of mitosis.
Table 1. Data for mitotic index study of cycloheximide treated maize
|Parameters||Control||60 min. @ 7.5 µg/ml||120 min. @ 7.5 µg/ml||60 min. @ 75.0 µg/ml|
|Maximum mitotic index:|
|Time (min) from treatment start||60||60||60||15|
|Mitotic index ± S.E.||7.02 ± 0.14||8.73 ± 0.62||8.73 ± 0.62||8.82 ± 0.21|
|% Metaphase||1.06||2.99||2.99||2.16 (2.75 @ 60')|
|% Anaphase + Telophase||1.52||0.07||0.07||1.72 (0.05 @ 60')|
|Minimum mitotic index:|
|Time (min) from treatment start||30||150||270||180|
|Mitotic index ± S.E.||6.86 ± 0.20,||5.17 ± 0.77||3.39 ± 0.33||3.35 ± 0.14|
|% Anaphase + Telophase||2.01||0.61||0.16||0.28|
|Total recovery time (min)||-||210-240||540-570||540-600|
|Length of time before prophase
frequency begins to decrease (min)
|Length of time before prophase frequency reaches minimum (min)||-||60-90||90||60-90|
The difference in total recovery time may be due to the disruption of other metabolic processes such as respiration or protein synthesis.
The effects of a 60-minute cycloheximide treatment on 14C-leucine incorporation into proteins were studied at various concentrations of cycloheximide and the percent inhibition is shown in Table 2 for both total and nuclear proteins. Labelling for the last 30 minutes of the cycloheximide treatments with 5.0 µCi/ml of 14C-leucine was found to be suitable. The results indicate a dose-dependent inhibition of label incorporation into proteins. The apparent selective decrease of label incorporation into nuclear proteins at 7.5 µg/ml of cycloheximide suggests that this chemical either selectively inhibits the synthesis of these nuclear proteins or their accumulation in the nucleus following synthesis.
Table 2. Inhibition of 14C-leucine incorporation-into
proteins as a function of cycloheximide concentration.
|Cycloheximide Concentration (µg/ml)||Total Proteins||Nuclear Proteins|
When label incorporation into proteins was monitored under the conditions used in the M.I. studies, it was found that for 75.0 µg/ml of cycloheximide, there was a rapid drop in the incorporation rate in the first 30-60 minutes. Following removal of cycloheximide, recovery of 60-70% of the activity occurred rapidly (about 1 hr), with the remaining recovery taking considerably longer (6-8 hr). Since cycloheximide can inhibit amino acid biosynthesis as well as RNA synthesis, the long recovery period probably represents the time required for the production of these precursors and the re-establishment of normal protein synthetic rates.
An interesting feature noted at a cycloheximide concentration of 75.0 µg/ml, was an elevation in the frequency of variants associated with chromosome structure and organization. At least nine classes were observed in this study and these could be arranged into two groups as shown in Table 3. The frequencies of the variants are shown in Table 4 in which the values represent the mean percent of variants in all metaphases, anaphases and telophases for 3 root tips in each treatment (about 2500 nuclei per root tip). In all cases, the treatment values were significantly higher than the controls and appeared to increase with time of treatment.
Table 3. Classes of cytogenetic variants.
I. Variants of chromosome organization
1. Metaphase with distinct chromatid separation
2. Anaphase with visible chromatid separation
3. Metaphase with random despiralization
4. Chromosomes with apparent banding
II. Variants of nuclear organization
1. Anaphase with bridges
2. Anaphase with laggards
3. Micronuclei and multiple daughter nuclei
4. Circular metaphases
5. Polar metaphases
Table 4. Frequencies of cytogenetic variants (mean
% ± S.E.).
|I. Variants of chromosome organization|
|15||2.23 ± 0.41||12.09 ± 0.33|
|30||2.24 ± 0.47||19.58 ± 1.52|
|60||1.63 ± 0.29||23.64 ± 0.92|
|II. Variants of nuclear organization|
|15||0.90 ± 0.45||5.78 ± 0.87|
|30||0||11.87 ± 0.52|
|60||1.14 ± 0.64||13.41 ± 0.59|
It is evident from these studies that, shortly after cycloheximide treatment, the frequency of cytogenetic variants increases. Since proteins are involved in the processes of chromosome condensation, movement and organization within the nucleus, the apparent decrease in proteins in the nuclear fraction following cycloheximide treatment may be responsible for the increased variants. An attempt to demonstrate an involvement of decreased nuclear protein levels with enhanced cytogenetic anomalies is presently underway.
C. L. Baszczynski, D. B. Walden and B. G. Atkinson
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