Tritiated Actinomycin D as a Cytochemical Label for Small Amounts of Dna

Actinomycin D blocks DNA-dependent RNA synthesis by binding to DNA (Reich et al., reference 15). On a cellular level, several radioautographic studies have demonstrated the in vivo binding sites of radioactive actinomycin (de Vitry, reference 19; Fraccaro et al., reference 3; Goldstein et al., reference 8; Ro et al., reference 16; Rothstein et al., reference 17; Sonneborn and Rothstein, reference 18). Brachet and Ficq (2) used actinomycin-1 C as a cytochemical label for DNA. The commercial availability of tritiated actinomycin D of high specific activity has raised the possibility of extending the sensitivity of this technique. Such an extension would have particular value at the present time when the question of endogenous DNA within organelles is of great interest. The present report concerns the amphibian oocyte nucleus. These nuclei contain approximately 1,000 nucleoli which are unattached to the chromosomes. Each of these nucleoli has been shown by two methods to contain a small amount of DNA: (a) under certain conditions the nucleoli are in the form of rings which can be fragmented by DNase (Miller, reference 13; Kezer, reference 10) (b) a Feulgen-positive granule is sometimes seen in association with each intact nucleolus (Brachet, reference ; Painter and Taylor, reference 14; Macgregor, reference 12; Gall, personal communication). However, the Feulgen reaction in these particles is usually quite weak, and during some stages of oogenesis staining is undetectable. For this reason, actinomycin D-3H was used as an alternate "stain" for nucleolar DNA. MATERIALS AND METHODS Actinomycin DH at a specific activity of 3.8-4.2 c/mmole was obtained from Schwarz Bio Research Inc., Orangeburg, N.J. Oocytes were obtained from newts of the species Triturus iridescens or Triturus cristatus carnifex for the preparation of chromosomes and nucleoli. Only mature oocytes were used for the preparations demonstrating localization of label in nucleoli. So that the number of mature oocytes in the ovary could be increased, a female was given three injections, over a week's time, of 100 IU of chorionic gonadotropin (Antuitrin S from Parke, Davis & Co., Detroit, Mich.) The oocytes were removed from the anesthetized animal, and the nuclei were isolated in 0.1 M "5:1" (5 parts 0.1 M KCI: 1 part 0.1 M NaCI). The nuclei were then transferred to well slides constructed as described by Gall (6). The well chamber had been previously washed with an aqueous solution of cold actinomycinD (Merck, Sharp & Dohme, West Point, Pa.; 10 pg/ml). This step is essential for reducing the background of the radioautographs to a reasonable level, since actinomycin binds strongly to glass. A drop of actinomycin D-3H was placed in the well chamber at a concentration of 30 uc/ml (about 10 p/g/ml) in 0.1 M 5:1. The isolated nucleus was transferred into the well, and the nuclear membrane was removed with forceps. The chromosomes and nucleoli were allowed to spread in the actinomycin D-3H solution for 1-3 hr at 4C. After this time, they were fixed to the bottom of the well chamber by exposing the preparation to the vapor of 50% ethanol. After fixation the actinomycin solution was washed out by placing the well slide into 0.05 M phosphate buffer, pH 6.8, for 5 min and then into water for 5 min. The preparation was then air dried. After the well slides had been separated from the slides bearing the chromosomes and nucleoli, the latter were washed in toluene and acetone and then