THE ORGANIZATION AND DUPLICATION OF CHROMOSOMES AS REVEALED BY AUTORADIOGRAPHIC STUDIES USING TRITIUM-LABELED THYMIDINEE

Information on the macromolecular organization of chromosomes and their mode of duplication has been difficult to obtain in spite of numerous attempts. One point of attack, long recognized but until recently unattainable, was the selective labeling of some component of the chromosome, the distribution of which could be seen in succeeding cell divisions. Reichard and Estborn' demonstrated that N15-labeled thymidine was a precursor of deoxyribonucleic acid (DNA) and that it was not diverted to the synthesis of ribonucleic acid. Recently Friedkin et al.2 and Downing and Schweigerl have used C'4-labeled thymidine to study DNA synthesis. In chick embryos and Lactobacillus there was no appreciable diversion of the tracer to ribonucleic acid. In view of these findings, thymidine appeared to be the intermediate required for the experiment, but the labels so far employed have not been satisfactory for microscopic visualization by autoradiographic means. In order to determine whether an individual chromosome among several in a cell is radioactive, autoradiographs with resolution to chromosomal dimensions must be obtained. Resolution at this level is difficult if not impossible to obtain with most isotopes, since the range of their beta particles is relatively great. Theoretically tritium should provide the highest resolution obtainable, since the beta particles have a maximum energy of only 18 Kev, corresponding to a range of little more than a micron in photographic emulsions. Consequently, identification of this label in particles as small as individual chromosomes should be possible. With this in mind; tritium-labeled thymidine was prepared and used to label chromosomes and to follow their distribution in later divisions by the use of photographic emulsions. Materials and Methods.-Tritium-labeled thymidine of high specific activity (3 X 101 mc/mM) was prepared by catalytic exchange of tritium from the carboxyl group of acetic acid to a carbon atom in the pyrimidine ring of thymidine (details of the method to be described elsewhere). Seedlings of Vicia faba (English broad bean) were grown in a mineral nutrient solution containing 2-3 jug/ml of the radioactive thymidine. This plant was selected because it has 121arge chromosomes, one pair of which is morphologically distinct, and because the length of the division cycle and the time of DNA synhesisl in the cycle are known.4 After growth of the seedlings in the isotope solution for the appropriate time, the roots were thoroughly washed with water and the seedlings were transferred to a nonradioactive mineral solution containing colchicine (500 jug/ml) for further growth. At appropriate intervals mots were fixed in ethanol-acetic acid (3:1), hydrolyzed 5 minutes in 1 N HC1, stained by the Feulgen reaction, 'and squashed on microscope slides. Stripping film was applied, and autoradiographs were prepared as described previously.5