Hybridization of Chromosomal Rna to Native Dna

Abstract

and Summary.-Chromosomal RNA, which is associated with chromosomal proteins in the chromosomes of higher organisms, possesses the ability to hybridize to homologous native DNA. The proportion of native DNA thus hybridized is similar to the proportion of denatured DNA which hybridizes with chromosomal RNA, and both are similar to the proportions of chromosomal RNA and DNA in native chromatin.We have found that chromosomal RNA possesses the ability to hybridize to homologous native DNA. Chromosomal RNA is a particular class of RNA distinguished by short chain length (40-60 nucleotides in length in different organisms),1' 2 high (8-10 mole %) content of dihydroprimidine (dihydrouridylic acid in pea, cow, and chick, dihydroribothymidylic acid in rat),3 and by its association with DNA in the chromosomes of higher organisms. In such chromosomes, chromosomal RNA is on the one hand bound to DNA in RNase-resistant form,4 and on the other is bound covalently to chromosomal protein. 3 We have previously shown that chromosomal RNA is sequence-heterogeneous and hybridizes to the extent of 3-5 per cent to homologous denatured DNA, the exact percentage depending upon the organism involved.2' 6 We show below that chromosomal RNA hybridizes to this same extent with homologous native DNA.Materials and Methods.-Preparation of chromatin: All of the experiments reported below were done with chromosomal RNA of rat Novikoff ascites tumor. The chromatin was prepared from tumor cells by the method of Dahmus and McConnell.2 The ascites cells were removed from the rat on the fifth to sixth day after infection with the tumor and were immediately washed by pelleting for 6 min at 700 g in TNKM buffer (0.05 M tris, pH 6.7, 0.13 M NaCl, 0.025 M KCl, 0.0025 M MgCl2), followed by a single pelleting from 4X-diluted TNKM buffer. The cells were then lysed in deionized water and the nuclei pelleted at 1500 g for 15 min. The nuclear pellet was homogenized by hand in a Teflon homogenizer and in 0.01 M tris, pH 8.0. The chromatin was pelleted and washed by repelleting four times from the same buffer. The resulting crude chromatin was then purified by layering it on 1.7 M sucrose and pelleting it for 2 hr at 22,000 rpm in the Spinco SW25 rotor.Preparation of chromosomal RNA: The sucrose density gradient-purified chromatin, dialyzed against 0.01 M Tris to free it of sucrose, was dissolved in 4 M CsCl and centrifuged for 18 hr at 35,000 rpm in the Spinco no. 40 rotor. Under these conditions, DNA and messenger RNA pellet, while chromosomal proteins, together with chromosomal RNA, float as a skin or pellicle.6 The chromosomal protein-RNA skin was next washed free of CsCl by pelleting it from 70% EtOH, suspended in 0.01 All tris pH 8.0, and protein-digested at 370C with preincubated (90 min, 370C) pronase, final concentration 1 mg/ml, for 4 hr at 370C. Any undigested and aggregated protein was centrifuged off; the RNA phenol-extracted in the cold and then precipitated from 0.2 N KAc by addition of 2 vol of EtOH. The RNA, redissolved in 7 Al urea co...

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