Rapidly labeled, polyribosome-associated RNA having the properties of histone messenger.

Histone synthesis and DNA replication (S phase) begin simultaneously in the HeLa cell life cycle. While these macromolecules are made, the cytoplasm contains a class of small polyribosomes which are absent in postmitotic (G,) cells and which disappear following exposure to cytosine arabinoside, an effective inhibitor of both DNA and histone synthesis. These polyribosomes incorporate a relative excess of lysine over tryptophan into their nascent polypeptides, supporting the hypothesis that they are the cytoplasmic site of histone synthesis.' The present report describes: (a) the isolation of both histone-like polypeptides and 7-9S RNA species from these polyribosomes, and (b) the relationship between this 7-9S RNA and histone synthesis with specific reference to the possibility that it is messenger RNA.2 Materials and Methods.-IMost of the techniques used in the present work have been described in previous publications. These include maintenance of HeLa S3 cells in suspension culture,3 preparation of cytoplasmic polyribosomes with the detergent Nonidet P40 (NP40),4 analysis of polyribosomes in sucrose gradients,5 and extraction of histones and their separation into different electrophoretic groups on acrylamide gels.' Cell synchronization: Although cells were usually synchronized by selective detachment of mitotic cells from monolayers,3 such populations become asynchronous late in the DNA-synthetic (S) and premitotic (G2) phases of the cell life cycle. For studies at those times, cells were synchronized by treatment in suspension with 2 mM thymidine6 for 16 hours, resuspension in fresh medium for 8 hours, followed by a second exposure to the drug for 16 hours. After a second resuspension in fresh medium, 90 per cent of the cells divided within 9 to 12 hours. Identification of polyribosome-associated polypeptides: To label nascent polypeptides with nearly equivalent amounts of radioactivity from each amino acid, 108 S-phase cells, resuspended in 10 ml of tryptophan, lysine, and serum-free growth medium,3 were incubated for one minute with 50,c C'4-lysine and 500 ,uc H3tryptophan. Cytoplasmic extracts were prepared with NP40,4 centrifuged through sucrose gradients, and analyzed for optical density at 260 mu and for acid-precipitable radioactivity.3 Appropriate fractions from the gradients were pooled, and polyribosomes were pelleted at 104,000 X g for one hour. Nascent polypeptides were released by treatment with 100 Mg/ml of ribonuclease at 37°C .7 The samples were dissolved in 1% sodium dodecyl sulfate (SDS), 0.5 M urea, 0.1% 2-mercaptoethanol (ME); dialyzed against 0.1% SDS, 0.1% ME in 0.01 11 phosphate at pH 7.4; and electrophoresed On 7.5% acrylamide gels.' G,and S-phase cells pretreated for one hour with 40 ug/ml cytosine arabinoside were similarly processed. Analysis of rapidly labeled, polyribosom.e-associated RNA: Synchronized cells at