Nucleic Acids Research: VOLUME 39 ISSUE 10 2011

Polysomal and nuclear poly(A)-containing RNA of normal rat liver and Novikoff hepatoma cells have been compared by cDNA.RNA hybridization kinetics. Homologous hybridization reactions revealed a total kinetic complexity of about 1.6x1O and 1.38x 1O daltons for liver and Novikoff mRNA respectively. The high abundance component present in liver cannot be detected in Novikoff. It was found from heterologous reactions that about 3O% by weight of mRNA sequences are specific to liver. Determination of the nuclear poly(A)-containing RNA complexities revealed that about 5.5% and 4% of the haploid genome is expressed in the liver and Novikoff respectively. In a heterologous reaction, up to 30% of the liver cDNA failed to form hybrids with Novikoff nuclear RNA. Cross hybridizations have further revealed abundance shifts in both nuclear and polysomal RNA populations. Some sequences abundant in liver are less abundant in Novikoff and some rare liver sequences are relatively abundant in Novikoff. INTRODUCTION Variation in gene expression occurs during development, in various differentiated tissues, as well as in response to various environmental stimuli. Many efforts have been made to elucidate the mechanisms involved in the regulation of gene expression. Comparison of the RNA populations of different cell types by molecular hybridization techniques has shown that diverse sets of sequences are transcribed in each differentiated cell type, providing strong evidence for transcriptional regulation of gene expression (1-3). Furthermore, it has been found, also by RNADNA hybridization, that nuclear RNA has a complexity which is several fold higher than cytoplasmic RNA (4-6). This finding, in combination with the existing evidence that mRNA is derived from larger nuclear precursors (7-9) suggests that the production of a mature mRNA molecule also involves a series of posttranscriptional events. © IRL Press Limited. 1 Falconberg Court. London W1V 5FG. U.K. 3193 Nucleic Acids Research It has been shown that tumor cells exhibit a number of changes from the normal phenotype which may be the result of specific alterations in the pattern of gene expression, leading to changes in the mRNA populations. Distortion of the control of gene expression at a posttrancriptional level has already been reported in animals fed carcinogenic amino azodyes (10-12) and also in some hepatomas (13-17). Other investigators have detected extensive elimination of certain RNA sequences from the nucleus of hepatomas (15,18,19). Since these results have been obtained from competitive hybridization experiments carried out under conditions which can detect differences only in the RNA copies of repetitive DNA sequences, they cannot be considered as completely representative of all sequence changes which have taken place. Our present results provide data concerned with the determination of the level and the extent of the possible alterations in gene expression between normal liver and hepatoma cells. In particular, we have analysed and compared the sequence complexity and the frequency distribution of poly(A)-containing RNA populations in normal liver and Novikoff hepatoma, since a number of differences have already been demonstrated at the protein level (20-22). We have studied the kinetics of homologous and heterologous RNA-cDNA hybridization reactions involving poly(A)containing RNAs from both cytoplasm and nucleus of normal liver and Novikoff hepatoma. We present evidence for restricted gene expression in the hepatoma as well as alterations in the relative abundances of different RNA classes in both nuclear and polysomal poly(A)-containing RNA populations. MATERIALS AND METHODS Animals and Tumor. Male Sprague Dawley rats (150—2OO g), used throughout, were fed ad libitum and kept in temperature-controlled rooms with alternating 12h-cycles of light and dark. Novikoff hepatoma was serially passaged in the same rat strain. Preparation of Polysomes. Polysomes were prepared from both rat liver and Novikoff hepatoma by a method described previously (23), as modified by Sippel et al. (24). To minimize possible contamination with nuclear RNP particles (25), the polysomes were frac-