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The 40s and 60s ribosomal subunits of L cells are y-irradiated in the absence of oxygen at low radiation doses to keep the integrity of the ribosomal structure. We show that under these experimental conditions, specific cross-links are induced in situ between rRNA and ribosomal proteins due to close contact between their reactive groups.We found that about 15 proteins are cross-linked to the 28s RNA. Most of them belong to the core proteins of the 60s ribosomal subunits. A few high-molecular mass proteins which might be factors are also bound to 28s RNA.Between 8 and 11 proteins are covalently linked to 18s RNA; 8 of these have been previously described as transferable proteins from one subunit to the other. Only 3 are core proteins of the small subunit.The contribution of these results to the study of the three-dimensional ribosomal structure is also discussed.The proteins and nucleic acids in ribosomes are arranged in a specific three-dimensional configuration to form structural and functional domains. Several studies have been made to identify the proteins that interact with the different species of MgSO,. Buffer I11 : 10 mM phosphate buffer pH 7.4, 0.5 mM MgSO,, 200 mM LiCl, 0.1 % sodium dodecylsulfate (SDS).Buffer IV: 10mM phosphate buffer pH 7.4, 100mM LiC1. rRNA~in eukaryotes [l, 21. During the last few years, covalent cross-linking techniques have been introduced for investigating the structural organisation of the ribosomes. Chemical reagents have been used in order to identify the binding sites for initiation factors [3,4] and to obtain information on the protein topography of both subunits [5 -71. Ultraviolet irradiation has been found to produce RNA-protein cross-links [8]. All these data are only now being integrated in terms of the ribosomal three-dimensional structure. Recently, y radiations appeared as an original and valuable tool to induce highly specific RNAprotein covalent bonds in Escherichia coli ribosomes [9,10]. At low doses, they reach the reactive targets in situ, while preserving the structure. Only a few chemical groups are capable of forming radicals and these reactive groups must be very close to form a covalent link [9, lo]. In order to gain more insight on the eukaryotic ribosomal structure, we applied, in this study, the y radiation technique to 40s and 60s ribosomal subunits of L cells. The proteins found cross-linked to rRNA have been identified among the distinct classes of ribosomal proteins defined in a previous study: those split off by KC1, core, transferable and labelled proteins [l 11.These results are discussed in a topological approach to the relationship of the RNA and the protein in the structure of the two ribosomal subunits. MATERIALS AND METHODS BuffersBuffer I contains 10 mM phosphate buffer pH 7.4, 5 mM MgSO,. Buffer I1 : 10 mM phosphate buffer pH 7.4, 0.5 mM Abbreviation. SDS, sodium dodecyl sulfate. Cell growth and labelling conditionsMouse C1 I D LM (TK-) cells were grown in suspension in an Eagle-Dulbecco medium supplemented with 10 % calf serum [12]. For la...

SummarySeparation of nuclear Hn-RNP particles into two density classes, following isopycnic centrifugation in metrizamide gradients, is reported. One class (density 1.31 g/ml) is rapidly labelled, and contains polydisperse heterogeneous high molecular weight particles, easily diffusible from intact nuclei under certain conditions. The other class (density 1.18 g/ml) is of lower molecular weight, not diffusible, and needs apparently a longer time to be labelled and/or to accumulate inside the nuclei.

Vero cells transfected with the S gene encoding the surface antigen (HBsAg) of the hepatitis B virus (HBV) synthesize HBsAg at low levels. We have obtained a large increase in S gene expression by somatic hybridization of Vero cells with primary hepatocytes, which are the natural target cells for HBV infection. Fusion with cells other than hepatocytes did not enhance expression of the S gene. The Vero/hepatocyte hybrid clones analyzed are stable and have maintained a high level of HBsAg synthesis over prolonged periods. Hybrid cell lines may be of general interest for the high-level synthesis of proteins using cloned genes.

Heterogeneous nuclear ribonucleoprotein particles (HnRNP) were separated in metrizamide density gradients, into two fractions migrating to 1.31 g ml-1 and 1.18 g ml-1, respectively. Proteins associated with each of these fractions were analysed by SDS-acrylamide gel electrophoresis. It is shown that the whole proteins extracted from these two metrizamide fractions exhibit clearly different electrophoretic patterns: 1.31 HnRNP particles contain as major polypeptide chains molecules with molecular weights ranging from 40,000 to 65,000, while major polypeptides of 1.18 HnRNP are banding in the 30,000-40,000 molecular weight region of the gels. Both fractions contain numerous other associated polypeptide chains whose molecular weights are above 65,000. A possible kinetic relationship between these two HnRNP classes was investigated in vivo by performing chase experiments. No clear evidence for a precursor-product relationship was found. Implications arising from these structural and kinetic observations, and problems relating to nuclear maturation of pre-messenger RNA, are discussed.

We report here data on the spontaneous resumption of proliferation in long-term primary cultures and we show that the proliferating areas are neoplastic. Normal rat hepatocytes were explanted in serum-supplemented Ham F12 medium and maintained over 8 months without transfer. The cells remained quiescent for the first 10 weeks and they were not tumorigenic when injected into nude mice. Later, without any modification of the culture conditions or transfer, progressive changes spontaneously occurred. Foci of dividing cells were detected, some displaying gamma-glutamyl-transpeptidase (gamma-GT) activity and F-actin fragmentation. These proliferating foci overcame the quiescent population. When injected into nude mice, the 15-week-old primary cultures were highly tumorigenic, with a 3-6 week latency for tumour formation. Furthermore, a cell line was derived from a primary culture started with a liver carcinogen promoter (biliverdin-enriched medium). This cell line proliferated rapidly and differed from a liver epithelial line, also established from our primary cultures, in its 1 karyotype (hyperploidy and translocation on chromosome 3), 2 requirement for arginine to proliferate, 3 gamma-GT positive reaction correlated to changes in actin fibre pattern, 4 sensitivity to protease inhibitors (i.e. alpha2 macroglobulin, PMSF) and 5 tumorigenicity. Long-term primary cultures and the karyotypically defined cell line are useful tools for further studies on in vitro genetic deviations.

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