DURATION OF THE NUCLEAR CYC LE IN TRADESCANTIA PALUDOSA ROOT TIPS AS MEASURED WITH H3-THYMIDINE

WIMBER, DONALD E. (Brookhaven National Lab., Upton, N. Y.) Duration of the nuclear cycle in Tradescantia paludosa root tips as measured with H3-thymidine. Amer. Jour. Bot. 47(10): 828-834. Illus. 1960. -The duration of the nuclear cycle and its various subdivisions were measured in Tradescantia root tips by autoradiographic techniques. H3-thymidine was used as a nuclear label and was supplied to the roots for 0.5 hr. After labeling, the roots were allowed to grow in the absence of label for periods up to 38 hr. By determining the percentage of divisions labeled at the various times of fixation, a reconstruction of the nuclear cycle could be made. The average cycle was determined as 20 hr. in duration, DNA synthesis 10.8 hr. , presynthetic interphase 4 hr. , postsynthetic interphase 2.7 hr. , prophase 1.6 hr. , metaphase 0.3 hr. and anaphase-telophase 0.6 hr. Approximate standard deviations for the duration of some of the subdivisions were calculated. THE STUDY of cell population kinetics has been simplified in recent years by the use of isotopically labeled substances that are utilized by cells in various synthetic activities. If an isotope is bound firmly within the cell, such labeled cells may be followed through one or more mitotic cycles by using microautoradiographic techniques. One of the most useful labeling substances in this regard is tritium-labeled thymidine, for it is incorporated primarily into deoxyribonucleic acid (DNA) with little or no turnover. Recently, cell renewal and generation time have been investigated with this isotope in a number of tissues and organisms. Cell population kinetics in blood and blood-forming organs in mammals have been one of the primary areas of investigation. Numerous papers in this field have been published in the last 3 years, some of the more recent of which are those by Cronkite et al. (1959), Bond et al. (1959), Lajtha (1959), Patt and Maloney (1959), and Schooley et al. (1959). Cellular proliferation in other tissues also has been investigated in detail. Reports of these studies have been made by Hughes (1958) and Hughes et al. (1958) who examined cell turnover in several tissues of the mouse. In the same manner Leblond et al. (1959) studied H3-thymidine uptake in tissues of the rat. Cellular proliferation and migration have been examined in the intestinal epithelium in particular by Quastler and Sherman (1959) in mice. In-vitro studies of cultures of human cancer cells have been made by Painter and Drew (1959) and Painter and Robertson (1959). A listing of many of the publications dealing with the use of H3-thymidine in the study of cell population kinetics may be seen in the 1 Received for publication February 12, 1960. This study was carried out at Brookhaven National Laboratory while the author was a Public Health Research Fellow (CF-8465) of the National Cancer Institute. The author gratefully acknowledges the advice of Drs. A. H. Sparrow and H. Quastler during the course of the work and discussions with Drs. P. S. Woods and D. Steffensen during the preparation of the manuscript. Appreciation is expressed to Miss Lillian Yu for technical assistance. bibliography on autoradiography prepared by Lima-de-Faria (1959). Work with isotopes along these lines in plants has been restricted mostly to that of Howard and Pelc (1951a, b; 1953) and of Howard (1956) who utilized p32 incorporation into root tip nuclei of Vicia faba as a cell-labeling device. Their results point to an average nuclear cycle 30 hr. in duration, about 6 hr. of which are taken up with DNA synthesis, 12 hr. with presynthetic interphase, 8 hr. with postsynthetic interphase and 4 hr. with mitosis. Siskin (1959) reported that the postsynthetic interphase in Tradescantia paludosa takes about 8 hr. and that the period from prophase to telophase takes approximately 2 hr. Two isotopic techniques have been employed in the study of cell turnoyer and generation time and may be categorized as the continuous and flash labeling methods. The first of these is exemplified in the studies of Howard and Pelc (1953). They exposed growing root tips to solutions containing radioactive phosphorus for varying periods up to 48 hr. ; autoradiographs of these root tips were prepared and processed in such a manner that presumably only the p32 incorporated into DNA in the nuclei remained. The total number of labeled nuclei and the number of mitotic nuclei registering positive autoradiographs were determined at each time period. An estimate of the total generation time is made from the rate of increase of the total number of labeled nuclei. The fraction of the generation time during which there is DNA synthesis equals the ratio of the percentage of total cells labeled after the tissue has been in contact with the isotope for a short time, over the percentage of cells which are labeled after a long exposure. The duration of the interval between synthesis and mitosis is deduced by noting the time of appearance of labeled dividing cells. This continuous-labeling technique is rather inexact, however, because the rate of increase of total number of labeled nuclei is not the best method for determining the total generation time. In addition, in-