Polyadenylated RNA Populations Research Articles

Major Temporal and Spatial Patterns of Gene Expression during Differentiation of the Sea Urchin Embryo

We have investigated the temporal and spatial patterns of accumulation of mRNAs randomly selected from the sea urchin gastrula polyadenylated RNA population. Three different assays show that the predominant temporal pattern of expression, exhibited by about three-fourths of these messages, consists of a large (mean 80-fold) increase in mRNA abundance between egg and gastrula stages. Most mRNAs are present in the maternal population and are detectable on blots as single mature-sized messages; however, a large number of high-molecular-weight, heterodisperse transcripts containing these same sequences also exist in the egg cytoplasm. The majority of gastrula messages are not embryo specific but are present in total adult urchin RNA at concentrations similar to those in embryos. Fine-scale RNA blot analysis indicates that the majority of mRNAs begin to accumulate at very early blastula stages, although there is considerable diversity in the time when these messages reach peak abundance. Most gastrula mRNAs are also spatially regulated during development. The observed distributions can be categorized into three major functional or regulatory classes: (1) Forty percent of mRNAs accumulate in cells which are cycling or preparing for growth. (2) About one-third of the messages accumulate in one or more differentiating cell types. (3) Only slightly more than one-fourth of the messages are present in all cell types throughout development. Most tissue-specific messages are relatively abundant, indicating that the differentiated functions of cells are executed through mRNAs operating at the level of hundreds of copies per cell. In contrast, most rare messages are expressed in most or all cell types, in which they function at only a few copies per cell. All messages which begin to accumulate before hatching blastula stage are initially distributed broadly, and their distribution becomes progressively restricted during embryogenesis. In contrast, all messages which begin to accumulate after the onset of gastrulation accumulate only in discrete subsets of cells. The results presented here illustrate much more extensive temporal regulation of gene expression during sea urchin embryogenesis than previously detected. This is accompanied by spatial regulation of expression of most genes which is itself temporally modulated as the cellular requirements for cell division and differentiation change during development.

Changes in the polyadenylated messenger RNA population during differentiation of Vicia faba root cells

Using cDNA probes, the polysomal polyadenylated RNA populations of Vicia faba meristematic, elongating, and mature root cells were analysed and compared, with respect to complexity, abundance distribution, and sequence representation. These cells express 12 300, 15 800, and 15 200 sequences, respectively, of an average size of 1300 nucleotides, distributed in three frequency classes. Transition from the meristematic to the elongating stage is coordinated with the disappearance of 25–30% of the abundant RNA species (2000 copies per cell) and with the appearance of new transcripts corresponding to 3000–3500 genes expressed at an intermediate level (50–60 copies per cell) and belonging to the rare class (about 4 copies per cell). These new transcripts represent 12.5% of the mass of the mRNA during the elongating stage and are quantitatively modulated during the transition to the mature stage. Thus 80 to 85% of the polysomal polyadenylated RNA species expressed in elongating or mature cells are common to the three cell types.

Polyadenylated RNA population present in dormant spores of Dictyostelium discoideum

Total RNA has been isolated from dormant spores of Dictyostelium discoideum. Although the amount of RNA per cell is smaller in spores than in growing amoebae, the ratio of poly(A) sequences to total RNA remains similar. Diversity and base sequence complexity of the polyadenylated RNA population have been examined by molecular hybridization with complementary DNA primed with oligo(dT). By this technique, the number of RNA species detected at more than one copy per cell is approximately 3000. RNA species can be classified in three sets of relative abundance, corresponding respectively to species present on the average at 1000 copies, 50 and four copies per cell. By heterologous hybridization it is shown that a large number of RNA species in spores are the same as those found at other stages of the cell cycle, while 20–30% of the RNA by mass appears specific to the spore cell. The specificity of the spore RNA population resides in the specific accumulation of a small number of RNA species.

The appearance of new polyadenylated nuclear RNA sequences during rat-liver regeneration.

Polyadenylated RNA populations from normal and 16-hour regenerating rat-liver nuclei were compared by heterologous hybridisation reactions with cDNA and unique DNA probes. Whereas unique DNA hybridisations did not show differences between the RNA populations, comparisons by cDNA hybridisation showed that about 10--15% by weight of polyadenylated sequences present in the nuclei of 16-hour regenerating rat livers were not found in the polyadenylated nuclear RNA of normal rat livers. These regenerating-specific nuclear cDNA sequences were isolated and characterised; the experiments showed that the complexity of the new sequences was 1-2 x 10(7) nucleotides (equivalent to 5,000--10,000 RNA sequences of 2,000 nucleotides in length) and that they were probably not potential messenger RNA sequences.

Changes in the complexity of nuclear RNA during development of Dictyostelium discoideum

Saturation hybridization with a radioactive single-copy genomic DNA probe has been used to measure the complexity of the total whole cell polyadenylated RNA populations during development of the cellular slime mold Dictyostelium discoideum. The complexity of the polyadenylated nuclear RNA has been deduced by comparison of these numbers with our previous estimates of the complexity of the polyadenylated RNA in the cytoplasm and on polysomes. Assuming asymmetric transcription, 53 to 54% of the single-copy portion of the genome is expressed in polyadenylated RNA in growing and early aggregating cells. Of this only about one-third or 19% is expressed on polysomes as mRNA. By contrast, during late aggregation and culmination, after the time when tips have formed on the aggregates a substantially greater portion of the single-copy genome, 80%, is expressed in the total population of polyadenylated RNA. Again, however, only about one-third or 30% of the single-copy genome is expressed as mRNA. Thus throughout growth and development the complexity of the polyadenylated nuclear RNA is about one and one-half times the complexity of the corresponding mRNA populations. The RNA population from the late developing cells is composed of all those RNA species expressed during growth and early development plus additional RNA species whose expression is specific to late development. Sequences which comprise the majority of the mass of the new polyadenylated RNA transcripts which appear in the cytoplasm and on the polysomes late in aggregation are not detectable in nuclear RNA from growing cells. This suggests that their developmental expression is largely regulated at the level of transcription rather than by controls at the level of processing or transport from the nucleus.

Isolation and characterization of polyadenylated RNA species from sporulating cells of Bacillussubtilis

Summary The appearance of a population of polyadenylated RNA during sporulation of B. subtilis is reported. This poly(A) RNA was characterized by hybridization to [ 3 H]poly(U), by its ability to bind oligo(dT)-cellulose and by its resistance to ribonucleases A and T 1 . Most of the poly(A) sequences are located at the 3′ terminus of the RNA chains and are of about 160–180 nucleotides length. Little or no poly(A) RNA was found in vegetative cells or in stationary stage cells of an asporogenic mutant blocked at zero stage of sporulation process. These results strongly suggest that the polyadenylated RNA might play a role in the regulation of gene expression during sporulation.

Identification of mRNA in the Slime MoldPhysarum polycephalum

Using a differential extraction procedure which had previously been shown to yield one nucleic acid fraction enriched in cytoplasmic RNA and another enriched in nuclear RNA, we have been able to isolate two polyadenylated RNA populations from microplasmodia of Physarum polycephalum. The poly(A)-containing RNA from the cytoplasmic-enriched fraction accounts for approximately 1.2% of the cytoplasmic nucleic acid, has a number-average nucleotide size of 1339+/- 39 nucleotides, and has been shown, in a protein-synthesizing system in vitro, to be capable of directing the synthesis of peptides which have also been shown to be synthesized in vivo by microplasmodia. The poly(A)-containing RNA from the nuclear-enriched fraction has a number-average nucleotide size of 1533 +/- 104 nucleotides and represents a mixture of cytoplasmic and nuclear adenylated RNA molecules. Based upon these observations, we have identified the polyadenylated RNA isolated from the fraction enriched in cytoplasmic nuclei acid as Physarum poly(A)-containing messenger RNA.

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.

High diversity in the polyadenylated RNA populations of embryonic myoblasts

High diversity in the polyadenylated RNA populations of embryonic myoblasts

Change in transcription patterns during early development of the sea urchin

Complementary DNA (cDNA) probes complementary to polyadenylated mRNA and HnRNA from sea urchin blastulas were synthesized by reverse transcriptase to analyze changes in prevalent class RNA during early development. About 17% of A +HnRNA and 62% of A +mRNA is transcribed into the cDNA probes. As measured by RNA-excess hybridization, the complexity of cDNA transcribed from HnRNA was 4.1 ± 1 × 10 7 nucleotides, and the complexity of cDNA from mRNA was 6.0 ± 1.2 × 10 5 nucleotides. These values correspond to 12 to 20% and 2 to 3% of the total blastula HnRNA and mRNA complexity, respectively. The cDNA thus represents a limited subset of the total mRNA sequences but a substantial fraction of the HnRNA sequences. The cDNA probes were hybridized with a large excess of the corresponding polyadenylated RNA populations from gastrula and pluteus embryos. Plateau values appeared to be substantially lower for the heterologous reactions. The kinetics of the reactions indicate a two- to threefold reduction in abundance of HnRNA species that persist to gastrula and a further small reduction at pluteus. The abundance of the persistent mRNA transcripts decreases 20-fold at gastrula but remains constant thereafter.

Analysis of Xenopus laevis ovary and somatic cell polyadenylated RNA by molecular hybridization

The number of sequences represented in the polyadenylated RNA population of the Xenopus oocyte and the degree to which these sequences are specific for early development have been determined by RNA-cDNA hybridization. Approximately 20,000 different sequences are found in the immature oocyte and the mature oocyte, and these sequences are the same in both types of cells. Approximately 5% of these sequences are present at a 15-fold higher concentration than the other 95%. The percentage of nonrepeated nuclear DNA homologous to the polyadenylated RNA was also determined and was in agreement with the number of sequences determined by the cDNA measurement. In addition, the number of sequences present in the Xenopus laevis kidney tissue culture cell and in the swimming tadpole was measured; approximately 20,000 different sequences were present in both the tissue culture cell and in the tadpole. The majority of the sequences present in the oocyte are not specific for early development since they are shared with both the tissue culture cell and the swimming tadpole; however, some sequences appear to be greatly increased in relative abundance in the tissue culture cell and the tadpole as compared to the oocyte.