The experiments described here used the single cross hybrid Seneca 60, kindly provided by D.B. Walden, University of Western Ontario.
Seeds were surface-sterilized with 2% sodium hypochlorite and germinated aseptically in petri dishes at 20 C in darkness. When the seedlings were four days old the apical 1 mm of the tips of 3 cm-long roots were processed either as fibre autoradiographs for replicon measurements, or as squash preparations for cell cycle and DNA synthetic (S)-phase determinations.
For the fibre work the root tips were exposed to high specific activity (70-90 Ci mmol-1)-tritiated[methvl-3H]-thymidine (3H-TdR) at a concentration of 1m Ci ml-1 for 30, 45, 60, 90, 120, 150 or 180 min at 20 C. Root meristem nuclei were then isolated and digested with trypsin on subbed microscope slides at 37 C. Sarkosyl and EDTA were then added and the resultant slurry, containing naked DNA, was spread across the slide. Slides were dried overnight in formalin fumes, treated with ice-cold trichloracetic acid, dehydrated with alcohol, air dried and prepared as permanent autoradiographs using Ilford K2 photographic emulsion. The exposure time was two to four months. Labelled DNA spread in this way is represented by tandem arrays of silver grains running across the slide. Replicon size is estimated by measuring the distance from the gap between a pair of these arrays to the corresponding gap in an adjacent pair. The relationship between mean length of labelled DNA segment and exposure time to 3H-TdR gives an estimate of the rate of single replicon fork movement, and dividing average replicon size by twice the rate of fork movement gives the time required for a replicon to replicate its allotted DNA (Rs).
For root meristem cells of Seneca 60 at 20 C, modal replicon size was 15 to 20 µm (about 45 to 60 kb of DNA), the rate of single fork replication was 3.0 µm h-1 (approximately 9 kb h-1), and Rs was 2.7h.
For the cell cycle work, roots of four-day-old seedlings were exposed to: 3H-TdR (S.A. 5Ci mmol-1; concentration 1µCi ml-1) for 0.5h, then unlabelled TdR (10-5M) for 0.5h. Next they were grown on moistened petri dishes at 20 C in darkness. Roots were fixed in 3:1 (v/v) absolute ethanol: glacial acetic acid every 2h for 24h after the start of 3H-TdR exposure. The apical 1 mm of the root tips were processed as Feulgen-stained squash preparations. These were made into permanent autoradiographs. The exposure time was 14 days. In each preparation 100 mitoses were scored for the presence or absence of silver grains, and hence for incorporation of 3H-TdR, in a series of random transects across the slide. These data were plotted against time after the start of labelling to give a percentage labelled mitoses curve (PLM). The duration of S-phase (about 4.7h) was estimated as the interval between the 50% intercepts of the ascending and descending limbs of the first peak in the PLM curve minus the 0.5h labelling period. The duration of the cell cycle (about 12 hours) was estimated from the mid-point of the first peak in the PLM curve to the corresponding point on the second peak.
The mean time for a replicon to replicate its DNA (Rs), and the duration of S-phase (Ds), were used to calculate an Rs:Ds ratio, a parameter which is positively correlated with synchrony of replication activation within a genome. The Rs:Ds ratio of 0.60 for Seneca 60 is within the range of values known for angiosperms (i.e. 0.11 - 0.71), and close to the values estimated by us for some other cereals (e.g. barley - 0.55; and hexaploid triticale - 0.53).
D. Francis1, A.D. Kidd1 and M.D. Bennett
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