CHARACTERIZATION OF CENTROMERE ARRANGEMENTS AND TEST FOR RANDOM DISTRIBUTION IN G0, G1, S, G2, G1, AND EARLY S' PHASE IN HUMAN-LYMPHOCYTES

The arrangement of centromeres, cluster formation and association with the nucleolus and the nuclear membrane were characterized in human ly mphocytes during the course of interphase in a cell-phase-dependent ma nner. We evaluated 3893 cell nuclei categorized by five parameters. Th e centromeres were visualized by means of indirect immunofluorescent l abeling with anticentromere antibodies (ACA) contained in serum of pat ients with CREST syndrome. The cell nuclei were classified as G0, G1, S, G2, G1' and early S' phase by comparing microscopically identified groups of cell nuclei with flow cytometric determination of cell cycle stage of synchronized and unsynchronized lymphocyte cell cultures. Ba sed on a discrimination analysis, a program was devised that calculate d the probability for any cell nucleus belonging to the G0, G1, S, G2, G1' and early S' phase using only two microscopic parameters. Various characteristics were determined in the G0, S, and G2 stages. A transi tion stage to S phase within G1 was detected. This stage shows centrom ere arrangements not repeated in later cell cycles and which develop f rom the dissolution of centromere clusters in the periphery of the nuc leus during G0 and G1. S phase exhibits various non-random centromere arrangements and associations of centromeres with the nucleolus. G1' a nd early S' phase of the second cell cycle display no characteristic c entromere arrangement. The duplication of centromeres in G2 is asynchr onous in two phases. For all cell phases a test for random distributio n of the centromeres in the cell nucleus was performed. There is a dis tinct tendency for centromeres to be in a peripheral position during G 0 and G1; this tendency becomes weaker in S phase. Although the visual impression is a seemingly random distribution of centromeres in G2 an d G1', statistical analysis still demonstrates a significant deviation from random distribution in favor of a peripheral location. Only the early S phase of the second cell cycle shows no significant deviation from a random distribution.