PATTERN OF SEGREGATION OF LABELED DNA AT THE CHROMATID LEVEL OF CHROMOSOMES WITH DIFFUSE CENTROMERES

Abstract

After pulse labeling Luzula purpurea shoot meristems with3H-thymidine, the cell cycle and its subdivisions were determined autoradiographically using the labeled mitoses method. The cycle time was 20 hr. G1 = 4.3 hr; C = 9.5 hr; G2 = 2.9 hr; and M = 3.3 hr. In subsequent experiments seedlings were labeled for 4 to 5 hr, long enough to label the chromosomes' full length, eliminating problems associated with asynchronous DNA replication. The longer labeling period did not alter the cell cycle parameters.Cells were designated as being in the first or second division after labeling by the time from the midpoint of the labeling period. Seedlings were placed in 0.1% cycloheximide solution to cause sister chromatids in metaphase cells to separate from each other. Autoradiographs were then made to determine segregation of labeled DNA at the chromatid level. At the first division after labeling all chromatids appeared to be labeled. At the second division after labeling with 3H-thymidine the labeling pattern was consistent with semiconservative segregation of labeled DNA and with sister chromatid exchange.These results are discussed in relation to cytological and photometric studies on chromosome strandedness in Luzula and in other organisms with chromosomes with diffuse centromeres.