CHROMOSOME STRUCTURE IN <i>VICIA FABA</i> AND <i>NARCISSUS JONQUILLA</i> BY ³H-THYMIDINE AUTORADIOGRAPHY AND TRYPSIN DIGESTION

Abstract

Chromosomes treated with trypsin have been studied by several investigators in order to observe the structure of the chromosome as to its strandedness and replication. The fact that chromatids consist of at least two strands when trypsin is applied, is agreed by Trosko and Wolff (1965) and Ghosh and Ghosh (1970), the former authors observing still smaller units.Furthermore, Ghosh and Ghosh (1970) have incorporated 3H-thymidine before applying trypsin to study the replication of chromosomes in Vicia faba. They have concluded that the mode of chromatid replication is semi-conservative. However, opinions vary concerning this subject.The purpose of the present investigation is to study the distribution of 3H-thymidine and the changes which may occur in mitotic chromosomes and in the chromatin of interphase nuclei in V. faba and Narcissus jonquilla when digested with trypsin. MATERIALS AND METHODS Vicia faba L.When the lateral roots of V. faba were about 0.5 cm the beans were skewered with a stainless wire and grown in distilled water for 2-3 days till the roots were about 1 cm long.They were then immersed in a 3H-thymidine (TRA 61 Thymidine 6-T(n) Batch 135 obtained from Japan Radiochemical Assoc.; S.A. 5 Ci/mM) solution, which was diluted with distilled water to make up a concentration of 3H-thymidine 2,ctCi/ml, at 20°C for 8 hr. The 8 hr treatment was calculated to be al out hilf of the cell cycle of root tip cells in V. faba from the report of Webster and Davidson (1968). After treating with 3H-thymidine the roots were washed in several changes of distilled water and transferred to 0.01% colchicine solution for 8 hr. The meristematic regions of the roots were cut off and fixed in cold 2% neatral f ormalin for 1-2 hr. To observe the effect of trypsin digestion the method by Trosko and Wolff (1965) wc,s followed.Hcwever, in order to isolate the n'zclei and chromosomes two layers of Tetoron T-2000 (screen gauze) supplied by Teijin Co. were used as filters. When the slides were air-dried, they were hydrolyzed in 1 N HCl at 60°C for 20 min and stained with Feulgen's nuclear reaction.For autoradiographic observation stripping film (Fuji stripping film ET-2F) was applied on the dry, stained material adhered to the gelatin coated slide glass.This was stored in a tightly sealed container at 5°C for 2-3 weeks. These slides were then