lnt roduct ionThe 5 S ribosxnal RNA of human, mouse, rabbit and rat cells have the same nucleotide sequence [l-3] , although this differs considerably from that of I:: co/i 141. In order to detertnine the extent of homology among eukaryotes we have studied the 5 S RNA of Xenvplrs luevis, the South African toad. T, and pancreatic ribonuclease digests of uniformly "2P-labelled 5 S RNA were studied and the results allow a sequence to be derived which differs only in eight positions from the mammalian sequence. MethodsUniformly 32P-labelled 5 S RNA was prepared by growing a X. laevis cell line (originally derived from kidney, and a gift from Dr. D.D. Brown) in [32P]phosphate. Cells were grown for 48 hr at 3.5" on the surface of plastic bottles in Eagle's medium with 80% Hank's salts and supplemented with 10% heated foetal calf serum and 50 &i/ml [32P]phosphate. After harvesting by trypsinization, the 5 S RNA was purified from ribosomes as before [?-I. In one experiment cells were lysed in 0.5Rj Nonidet P40 in 0.01 M KCI, 0.001 M magnesium acetate, 0.01 M Trischloride, pH 7.5 for 10 min at 0". SDS was added to 0.2% and the RNA extracted by shaking with an equal volume of phenol for 1 hr at room temperature. 5 S RNA was purified by 1 CC%. acrylamide gel electrophoresis on slab gels [ 5 ] .

Details are presented of the in vitro synthesis of double-stranded DNA complementary to purified Xenopus globin messenger RNA, using a combination of reverse transcriptase, fragment 'A' of E. coli DNA polymerase 1 and S1 endonuclease. After selection of duplex DNA molecules approaching the length of Xenopus globin messenger RNA by sedimentation of the DNA through neutral sucrose gradients, the 3'-OH termini of the synthetic globin gene sequences were extended with short tracts of oligo dGMP using terminal transferase. This material was integrated into oligo dCMP-extended linear pCR1 plasmid DNA and amplified by transfection of E. coli. Plasmids carrying globin sequences were identified by hybridization of 32P-labelled globin mRNA to total cellular DNA in situ, by hybridization of purified plasmids to globin cDNA in solution, by analysis of recombinant DNA on polyacrylamide and agarose gels, and by heteroduplex mapping. The results show that extensive DNA copies of Xenopus globin mRNA have been integrated into recombinant plasmids.

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