Changes in nuclear and polysomal polyadenylated RNA sequences during rat-liver regeneration.

Nuclear and polysomal polyadenylated RNA populations of normal and 16 hour regenerating rat liver have been compared by mRNA-cDNA hybridisations and by unique DNA saturation experiments. It was found that nuclear polyadenylated RNA hybridises to 6.8% of unique DNA in both normal and 16 hour regenerating rat liver. However, cross-hybridisation experiments using cDNA have shown that 10-15% by weight of nuclear polyadenylated RNA sequences are specific to 16 hour regenerating rat-liver. Since both unique DNA and cDNA hybridisation have shown that normal and 16 hour regenerating rat-liver polysomal polyadenylated RNA populations are qualitatively very similar sequences specific to 16 hour regenerating rat-liver nuclear polyadenylated RNA are nucleus confined. Polysomal RNA sequences which were abundant in normal rat-liver have become less abundant in regenerating rat liver.     

Differences in abundance of individual RNAs in normal and animalized sea urchin embryos

A library of cDNA clones was constructed representing polysomal polyadenylated RNA of mesenchyme blastulae of Strongylocentrotus purpuratus. Using this library, we determined whether or not individual RNA species are associated with animalization of embryos by zinc ions. Clones corresponding to the most actively synthesized RNAs during the period just prior to the mesenchyme blastula stage were selected by screening colonies with in vivo-labeled RNA. The most abundant of these were chosen for further study. Individual RNA abundance was measured as percent of mass of total polyadenylated RNA by hybridizing cDNA exhaustively with cloned DNA on filters. The RNAs in the selected, cloned sequences were present in abundances of 0.01 to 1% of the mass of polyadenylated RNA. Changes in abundance of individual RNA species occurred during normal development and departures from these developmental changes occurred in the zinc-animalized embryos. Two RNA species, which normally increase 10-fold in abundance, are drastically repressed and at least one RNA species increases in abundance dramatically in the animalized embryos. These departures from the normal program of presumptive gene expression may furnish insights into changes in the normal processes of development.     

Frequency distribution of pre-messenger RNA sequences in polyadenylated and non-polyadenylated nuclear RNA from Friend cells.

Hybridisation of cDNA probes for abundant and rare polysomal polyadenylated RNAs with polyadenylated and non-polyadenylated nuclear RNA from Friend cells indicated that the abundant polysomal polyadenylated RNA sequences were present at a higher concentration in the nucleus than rare polysomal sequences, but at a reduced range of concentrations. The ratio of the concentrations of abundant and rare sequences was about 3 in non-polyadenylated nuclear RNA, 9 in polyadenylated nuclear RNA and 13 in polysomal polyadenylated RNA. This suggests that polyadenylation may play a role in the quantitative selection of sequences for transport to the cytoplasm. Polyadenylation cannot be the only signal for transport, since a highly complex population of nucleus-confined polyadenylated molecules exists, each of which is present on average at less than one copy per cell.     

Differences among the polyadenylated RNA sequences of human leucocyte populations: an approach to the objective classification of human leukaemias

We have constructed a complementary DNA (cDNA) library representing expressed sequences of the white blood cells from a patient with chronic granulocytic leukaemia. The library was screened by colony hybridization of 32P-labelled cDNAs synthesized from the polyadenylated RNAs of the white blood cells from patients with chronic granulocytic or chronic lymphocytic leukaemia. The autoradiographic patterns were compared and 70 recombinants were selected to comprise a panel which distinguished between these two types of leukaemia. Hybridization of this panel with complementary DNAs transcribed from the polyadenylated RNAs of a variety of normal and neoplastic leucocyte populations showed that the RNA sequences in high abundance in leucocytes from chronic granulocytic leukaemias differ quite radically from those in other leucocytes. The patterns of hybridization seen when this panel was challenged with cDNAs representing the RNAs of normal and leukaemic leucocyte populations were sufficiently different to distinguish clearly the peripheral blood leucocytes of chronic granulocytic leukaemias from other populations of white blood cells, both normal and leukaemic. We suggest that this approach might provide additional markers useful in the classification of the acute leukaemias, especially the undifferentiated leukaemias whose identification by conventional methods is uncertain.     

Frequency distribution of messenger sequences within polysomal mRNA and nuclear RNA from rat liver.

DNA complementary to polysomal poly(A)-containing mRNA (cDNA) of male rat liver was used to study the diversity of messenger sequences in the nucleus and in polysomes. 1. Hybridization of cDNA against an excess of its own polysomal mRNA template revealed that about 10,000 different mRNA species are expressed in the liver tissue. They are distributed in a wide frequency range derived from approximately 0.5% of the total genome. 2. Hybridization of the cDNA against total nuclear RNA shows that messenger sequences comprise less than 1% of the mass of total nuclear RNA. Messenger sequences have a different frequency distribution in nucleus and cytoplasm. 3. In hybridizations using cDNA, which had been fractionated into sequences representing abundant and scarce polysomal mRNA molecules, it was found that although abundant cytoplasmic messenger sequences are also abundant in the nucleus, they exist in a significantly lower frequency range in the nuclear compartment.     

Changes in the frequency and diversity of messenger RNA populations in the course of myogenic differentiation.

Complementary DNAs (cDNAs) were synthesized from polyadenylated RNAs of myoblasts and myotubes and used to analyze changes in the sequence complexity and frequency distribution of messenger RNAs during myogenesis in vitro. cDNA . polyadenylated-RNA hybridization kinetics show the presence of messenger RNA sequences specific for myotubes in fully differentiated muscle cultures. These sequences are accumulated just prior to fusion, as was shown by hybridizations of myotube cDNA and total cytoplasmic RNAs from cells at different stages of differentiation. The myotube cDNA can be enriched 10-fold in myotube-specific RNA species by a hybridization with cytoplasmic RNAs from myoblasts and subsequent removal of these hybridized sequences by hydroxyapatite.     

Comparison of complexity and diversity of polyadenylated polysomal and informosomal messenger ribonucleic acid from Chinese hamster cells.

The sequence complexity and relative abundance of cytoplasmic polyadenylated polysomal (ribosome-bound) mRNA and cytoplasmic polyadenylated informosomal (ribosome-free) mRNA were analyzed in exponentially growing Chinese hamster cells (line CHO) using the technique of cDNA hybridization to excess poly(A)+ mRNA. Polysomal and informosomal mRNAs had similar complexities ( approximately 8300 mRNA species), but both the fraction of mRNA and the number of sequences comprising the mRNA abundance classes were different. Heterologous annealing reactions showed that all of the mRNA sequences detected were shared by the polysomal and informosomal mRNAs. However, the most abundant informosomal mRNA component was considerably different from the most abundant polysomal mRNA component. For a more detailed analysis, cDNA complementary to the most abundant informosomal and polysomal mRNAs was isolated. By use of the fractionated cDNA, it could be demonstrated that the most abundant informosomal mRNA sequences were distributed in the polysomal mRNA with an approximately fivefold reduction in relative frequency. These results are not compatible with models postulating translational control of gene expression by the complete sequestering of some mRNA sequences in an untranslatable form in the cytoplasm. The data are, however, consistent with models encompassing differential rates of initiation on the polysome and/or preferential affinity of some mRNAs for initiation factors.     

Sequence complexity and tissue distribution of chick lens crystallin mRNAs

Using hybridization reactions with a cDNA copy, the complexity of polysomal polyadenylated mRNA from the day-old chick lens was found to correspond to 5800–7200 sequences of average size, arranged in three abundance classes. Experiments with heterologous cDNAs suggest on a qualitative basis that many of the sequences expressed in 8-day embryonic neural retina and pigmented epithelium mRNAs are also present in lens mRNA. A cDNA fraction complementary to the most abundant lens mRNAs, representing an approximate minimum of four sequences, was used to assay the dosage of putative crystallin sequences in these and other embryonic tissues. Neural retina and pigmented epithelium cytoplasmic mRNAs have low concentrations of these sequences, which appear to be absent from mRNA prepared from headless bodies and muscle.     

alpha-Fetoprotein and albumin mRNA levels in liver regeneration and carcinogenesis

Using a titration procedure, we measured the proportion of alpha-fetoprotein (AFP) and albumin mRNA in normal, regenerating, and preneoplastic rat livers. AFP mRNA constitutes approximately 0.006% of the polysomal polyadenylated RNA of normal livers and this proportion increases only slightly before the onset of DNA synthesis in liver regeneration induced by partial hepatectomy or CCl4 injury. In either model of liver regeneration, the proportion of AFP mRNA in polysomal RNA is highest approximately 24 h after the peak of DNA synthesis. The increase in the proportion of AFP mRNA in polysomal RNA is relatively small during liver regeneration (2-4-fold) but is larger (30-50-fold) in preneoplastic livers of rats fed a choline-deficient diet containing 0.1% ethionine. In contrast to those changes in AFP mRNA, albumin mRNA levels remain unchanged during liver regeneration and double in preneoplastic livers. Our results indicate that the concept of "retrodifferentiation" as it applies to liver regeneration and certain types of hepatic neoplasia needs reevaluation.     

Comparison of polysomal and nuclear poly(A)-containing RNA populations from normal rat liver and Novikoff hepatoma.

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 at total kinetic complexity of about 1.6 X 10(10) and 1.38 X 10(10) 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 30% 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.