Polyadenylated RNA isolated from the archaebacterium Halobacterium halobium.

Polyadenylated [poly(A)+] RNA has been isolated from the halophilic archaebacterium Halobacterium halobium by binding, at 4 degrees C, to oligo(dT)-cellulose. H. halobium contains approximately 12 times more poly(A) per unit of RNA than does the methanogenic archaebacterium Methanococcus vannielii. The 3' poly(A) tracts in poly(A)+ RNA molecules are approximately twice as long (average length of 20 nucleotides) in H. halobium as in M. vannielii. In both archaebacterial species, poly(A)+ RNAs are unstable.     

Polyadenylated, noncapped RNA from the archaebacterium Methanococcus vannielii.

Polyadenylated [poly(A)+] RNA molecules have been isolated from Methanococcus vannielii by oligodeoxythymidylate-cellulose affinity chromatography at 4 degrees C. Approximately 16% of the label in RNA isolated from cultures allowed to incorporate [3H]uridine for 3 min at 37 degrees C was poly(A)+ RNA. In contrast, less than 1% of the radioactivity in RNA labeled over a period of several generations was contained in poly(A)+ RNA molecules. Electrophoretic separation of poly(A)+ RNA molecules showed a heterogeneous population with mobilities indicative of sizes ranging from 900 to 3,000 bases in length. The population of poly(A)+ RNA molecules was found to have a half-life in vivo of approximately 12 min. Polyadenylate [poly(A)] tracts were isolated by digestion with RNase A and RNase T1 after 3' end labeling of the poly(A)+ RNA with RNA ligase. These radioactively labeled poly(A) oligonucleotides were shown by electrophoresis through DNA sequencing gels to average 10 bases in length, with major components of 5, 9, 10, 11, and 12 bases. The lengths of these poly(A) sequences are in agreement with estimates obtained from RNase A and RNase T1 digestions of [3H]adenine-labeled poly(A)+ RNA molecules. Poly(A)+ RNA molecules from M. vannielii were labeled at their 5' termini with T4 polynucleotide kinase after dephosphorylation with calf intestine alkaline phosphatase. Pretreatment of the RNA molecules with tobacco acid pyrophosphatase did not increase the amount of phosphate incorporated into poly(A)+ RNA molecules by polynucleotide kinase, indicating that the poly(A)+ RNA molecules did not have modified bases (caps) at their 5' termini. The relatively short poly(A) tracts, the lack of 5' cap structures, and the instability of the poly(A)+ RNA molecules isolated from M. vannielii indicate that these archaebacterial poly(A)+ RNAs more closely resemble eubacterial mRNAs than eucaryotic mRNAs.     

Polyadenylated RNA in two filamentous cyanobacteria.

Polyadenylated RNA was detected in the cyanobacteria Nostoc sp. strain MAC and Anabaena variabilis by oligodeoxythymidylic acid-cellulose chromatography and by hybridization to [3H]polyuridylic acid. Polyadenylate tracts from A. variabilis were located at the 3' end of RNA chains and had an estimated length of 15 to 22 nucleotides.     

Euglena gracilis Chloroplast DNA Codes for Polyadenylated RNA

Polyadenylated RNA, isolated from total cellular RNA of photoautotrophically grown Euglena gracilis, comprised 2.1% of the total cellular RNA and contained 6.2% polyadenylic acid. Polyadenylated RNA, labeled in vitro with ¹²⁵I, hybridized at saturating levels to an average 7.7% of the chloroplast DNA. In the presence of excess chloroplast rRNA, hybridization of polyadenylated RNA was reduced, but was still observed at a level corresponding to 2.8% of the chloroplast DNA. Polyadenylic acid was not detected in mRNA prepared from chloroplast polyribosomes, indicating a level of less than 0.1% polyadenylic acid in mature chloroplast mRNA. Of the total RNA isolated from cytoplasmic polyribosomes, 2.0% contained polyadenylic acid. This latter polyadenylated RNA did not hybridize to chloroplast DNA.     

Polyadenylated RNA in the lower eukaryote Physarum polycephalum.

Utilizing a method which quantitatively extracts high molecular weight RNA, including intact precursor as well as mature ribosomal RNAs, adenylated molecules have been isolated from nuclear- and cytoplasmic-enriched fractions of Physarum microplasmodia labeled with [3H]-uridine. Electrophoretic analysis of denatured adenylated RNA from the nuclear-enriched fraction indicated the presence of a population of large molecules not found in the cytoplasmic-enriched fraction.     

Polyadenylated RNA complementary to repetitive DNA in mouse L-cells.

Complementary DNA, synthesized with L-cell polyadenylated RNA as template, renatured with total L-cell DNA to about 70%. About 30% complementary to unique sequence DNA and another 10 and 30% corresponded to sequences about 20- and 500-fold repetitive. Complementary DNA was fractionated after partial hybridization with total polyadenylated RNA to obtain preparations enriched or impoverished in complements of the most frequent polyadenylated RNA. Renaturation of these complementary DNA fractions with L-cell DNA revealed that most frequent RNAs are transcribed from repetitive DNA sequences, Complementary DNA, density labeled with bromodeoxyuridine, was fractionated by renaturation with L-cell DNA to yield fractions enriched in repetitive and unique sequence DNA. The denisty labeled complementary DNA was purified by equilibrium centrifiguation in an alkaline Cs2SO4 gradient. The complementary DNA representing mainly repetitive DNA sequences hybridized preferentially to frequent polyadenylated RNA.     

Polyadenylated RNA of Volvox: isolation and partial characterization

Polyadenylated RNA from Volvox carteri has been isolated and partially characterized. Electrophoretic profiles of total cellular poly(A)-associated RNA of Volvox spheroids indicate a hetero-disperse distribution of size classes with the range extending from an apparent sedimentation value of approximately 10S to greater than 38S. The radioactive labelling kinetics of this material are typical for rapidly-turning-over RNA. The profiles of poly(A) RNA from different cell types show marked differences in average migration rate. Terminally-differentiated somatic cells contain a greater proportion of material of higher molecular weight than either gonidia (germ cells) or cleaving embryos. The poly(A) segments associated with cellular RNA, obtained by selective RNase digestion are heterogeneous in size as determined by gel electrophoresis with the largest tracts estimated to be 75-80 nucleotides long. Gonidia and embryos display the greatest degree of size heterogeneity, while somatic cells show predominantly the largest classes of poly(A) tract. It is apparent that gross changes in poly(A) RNA metabolism accompany development and cellular differentiation in Volvox.     

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.     

Polyadenylated and nonpolyadenylated messenger RNA fractions from sea urchin embryos code for the same abundant proteins.

RNA was extracted from polysomes of sea urchin mesenchyme blastulas and fractionated by affinity chromatography on oligo(dT)-cellulose. The poly(A)+ and poly(A)? fractions were translated in cell-free systems derived from wheat germ and rabbit reticulocytes. The translation products were analyzed by two-dimensional electrophoresis on polyacrylamide gels and found to be qualitatively similar for poly(A)+ and poly(A)? mRNA. Most of the products of cell-free translation have been identified among the in vivo translation products, indicating the fidelity of the translation systems. At least 85% of the poly(A)? mRNA lacks detectable (8 nucleotides or longer) tracts of poly(A). Less than 11% of the poly(A)? mRNA entering polysomes in the reticulocyte lysate contains detectable homopolymers of adenosine. We conclude that the poly(A)+ and poly(A)? mRNA code for the same set of abundant proteins, having isoelectric points between 5 and 7.2 and molecular weights between 15,000 and 100,000. It is possible that some proteins, such as histones, not detectable in our analysis are coded for exclusively by mRNA having or lacking poly(A) tracts.     

Intracellular transport of nuclear ribosomal RNA in Acetabularia mediterranea]

The ribosomal RNA transport from a nucleus to a perinuclear cytoplasm and its following distribution in the cytoplasm of Acetabularia mediterranea cells were studied using transplantation of RNA-labeled rhizoid into unlabeled stalk. In addition rifamycin treatment was used for inhibition of cytoplasmic RNA synthesis. Acetabularia nuclei contain the stable RNA fractions similar to those present in some other eukaryotes. Nuclear 25S and 17S ribosomal RNA rapidly enter the rhizoid cytoplasm whereas the following trasfer of them to other regions of the cell is a very slow process. Within two days only an insignificant part of 25S and 17S ribosomal RNA is transferred from the rhizoid to the stalk and is distributed there over the base-apical gradient. No preferential transfer of the nuclear ribosomal RNA to the apical region was observed.     

Gradient of RNA distribution in the cytoplasm of Acetabularia mediterranea

Radiochemical studies and electrophoresis showed that there exists an apico-basal gradient of RNA concentration in Acetabularia mediterranea cytoplasm. The main contribution to the formation of such a gradient is made by the different rates of RNA turnover in the cytoplasm rather than by the transfer of nuclear RNA. High molecular weight RNA fractions synthesized in the cytoplasm originate from chloroplast ribosomes; their sedimentation constants are close to those of 23S and 16S rRNA fractions of E. coli.