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intermediates in a process leading to the formation of 4S RNA, which, by virtue of a looser secondary structure, are less effectively retained by columns of 'Sephadex 0-100'. To evaluate this, a "chase" type of experiment was carried out. The tumour cells were labelled for 5 min, treated with actinomycin D (50 fJ-g/ml.) for 15 min to inhibit synthesis of 5S and 4S RNA, washed twice and finally suspended in fresh medium containing large amounts of non-radioactive uridine (0•1 mmolar), Lmethionine (0•1 mmolar), cytidine (1 mmolar) and sodium formate (20 mmolar) and incubated for a further 1 h. This "actinomycin-pulse" before the "chase" procedure is required with these tumour cells to obtain maximal blocking of RNA synthesis, because it is difficult to dilute the radioactivity in the nucleotide precursor pools sufficiently quickly. The result of this type of experiment is shown in Fig. 1d, and demonstrates that the level of tritium radioactivity eluting in the region corresponding to 4S RNA after the "chase" correlates well with a redistribution of the radioactivity which originally appeared, for example, in the C and D regions after 5 min of incubation (Fig. la). If the additional components (peaks C and D) represent intermediate steps of a process leading to the "maturation" of 4S RNA, the question arises as to whether or not a puromycin-sensitive step is involved, as seems to be the case in the process leading to the synthesis of ribosomal RNA from precursor RNA (refs. 6 and 7). When the incubation of the tumour cells was carried out in the presence of puromycin the "maturation" process appeared to be unaffected (Fig. la), although the radioactivity in the excluded ribosomal peak was reduced. It seemed also of interest to determine to what extent the process depended on methylation of RNA. Previous treatment of the tumour cells with DL-ethionine was sufficient to depress, by about 75 per cent, the subsequent incorporation of labelled methyl groups from labelled (methyl)-L-methionine into RNA. Presumably this was because enough S-adenosylethionine 8 • 9 was formed within the cells to impair either the formation, or functioning, of 8-adenosyl-L-methionine, the donor of methyl groups to RNA. The pattern of incorporation of label from tritiated uridine added after the preincubation period with DL-ethionine indicates that the "maturation" process was only slightly impaired, which suggests that, a lthough possibly a number of steps are involved in the biogenesis of 4S RNA in Krebs II ascites tumour cells, insertion of methyl groups appears to be a refinement rather than an obligatory step, although there is some evidence from bacterial systems that "methyl-deficient" transfer RNA has altered physical properties 10 • One of us (B. M. L.) is a Colombo Plan Fellow. 'Ve thank Professor J. N. Davidson for his interest and for providing the facilities with the aid of a grant from the British Empire Cancer Campaign for Research. We also thank Miss E. A. Jess for technical assistance.

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