Dna Synthesis and Chromosomal Asynchrony

A number of studies investigating the chronology of D N A synthesis in human chromosomes have indicated a degree of asynchrony among the members of the complement. The most striking asynchrony of D N A synthesis demonstrated within pairs of human chromosomes is the late replication of one of the X chromosomes shown by pulse labeling with tritiated thymidine in cultures of leucocytes (3, 4, 7, 8). Morishima et al. (8), have presented evidence that this late replicating X chromosome is the same chromosome which produces the sex chromatin body in interphase somatic nuclei of females. Their study covered the late S phase and did not determine whether the asynchrony occurred also at the beginning of replication of these chromosomes in the early S phase. In order to observe chromosomal synchrony or asynchrony in early S phase, it was useful to have a greater degree of synchrony of cell division than ordinarily is found in cultures of human leucocytes, and to know when a large number of cells in a culture could be labeled for autoradiography of the very beginning of D N A synthesis. It is known that aminopterin, a folic acid analog, acts as an antagonist of tetrahydrofolic acid in the biosynthesis of thymidylic acid, and thus inhibits D N A synthesis and subsequent mitosis (5). In the present study, using leucocytes from normal human females, the degree of parasynchrony of D N A synthesis in the culture was enhanced by a period of aminopterin inhibition followed by simultaneous release from inhibition and pulse labeling of D N A with tritiated thymidine in what is presumed to be, for most of the cells, the early S phase. A period of incubation allowed cells thus labeled to complete the S phase and G~ phase and proceed into mitosis. A high proportion of metaphases of such cells was found to have all chromosomes labeled except one, which by size and morphology was found to be in the 6 12 + X group (2), and is presumed to be the heteropycnotic X chromosome.