Terminal completion of poly(A) synthesis in sea urchin embryos.

The kinetics of accumulation of nuclear and cytoplasmic poly(A) have been determined in sea urchin blastulas and gastrulas, stages when essentially all mRNA is synthesized de novo in the nucleus. A majority of the labeled poly(A) is found in the cytoplasmic fraction after a brief pulse. The ratio of radioactive AMP to adenosine in pulse-labeled nuclear, cytoplasmic, and polyribosomal poly(A) is considerably less than the number average length of the labeled poly(A), indicating that there is 3′-terminal addition of adenosine to previously synthesized poly(A). The size distribution of pulse-labeled, terminally elongated poly(A) in the cytoplasm is similar to that of the largest nuclear poly(A) rather than the steady-state size distribution of cytoplasmic poly(A), which is smaller and more heterogeneous. The most likely interpretation of these results is that there is a predominant 3′ terminal addition of short tracts of adenosine to poly(A) attached to nuclear RNA just before or during entrance of this RNA into the cytoplasm. In this respect, much of the 3′ terminal addition may be thought of as terminal completion of poly(A) synthesis.     

Sequence-specific adenylations and deadenylations accompany changes in the translation of maternal messenger RNA after fertilization of Spisula oocytes

A dramatic change in the pattern of protein synthesis occurs within ten minutes after fertilization of Spisula oocytes. This change is regulated entirely at the translational level. We have used DNA clones complementary to five translationally regulated messenger RNAs to follow shifts in mRNA utilization at fertilization and to characterize alterations in mRNA structure that accompany switches in translational activity in vivo. Four of the mRNAs studied are translationally inactive in the oocyte. After fertilization two of these mRNAs are completely recruited onto polysomes, and two are partially recruited. All four of these mRNAs have very short poly(A) tracts in the oocyte; after fertilization the poly(A) tails lengthen considerably. In contrast, a fifth mRNA, that encoding alpha-tubulin mRNA, is translated very efficiently in the oocyte and is rapidly lost from polysomes after fertilization. Essentially all alpha-tubulin mRNA in the oocyte is poly(A)+ and a large portion of this mRNA undergoes complete deadenylation after fertilization. These results reveal a striking relationship between changes in adenylation and translational activity in vivo. This correlation is not perfect, however. Evidence for and against a direct role for polyadenylation in regulating these translational changes is discussed. Changes in poly(A) tails are the only alterations in mRNA sizes that we have been able to detect. This indicates that, at least for the mRNAs studied here, translational activation is not due to extensive processing of larger translationally incompetent precursors. We have also isolated several complementary DNA clones to RNAs encoded by the mitochondrial genome. Surprisingly, the poly(A) tracts of at least two of the mitochondrial RNAs also lengthen in response to fertilization.     

Nucleocytoplasmic transport of RNA. The effect of 3''-deoxyadenosine triphosphate on RNA release from isolated nuclei.

The addition of 3'-deoxyadenosine (cordycepin) to cells in culture results in the inhibition of the appearance of mRNA in the cytoplasm through a mechanism thought to involve the inhibition of polyadenylate synthesis. I studied the effect of 3'-deoxyadenosine triphosphate, the physiologically active form of 3'-deoxyadenosine, on RNA release from isolated nuclei. Nuclei were isolated from baby-hamster kidney (BHK) fibroblasts that had been given a short pulse of radioactive uridine or adenosine in the presence of a low concentration of actinomycin D before harvest. RNA release from the isolated nuclei under the appropriate incubation conditions was time-, temperature- and ATP-dependent. 3'-Deoxyadenosine triphosphate inhibited RNA release from the isolated nuclei. However, RNA that was restricted to the nuclei during incubation with the drug could be chased out of the nuclei if the incubation medium was replaced with medium containing only ATP. The chased poly(A)+ (polyadenylated) RNA had shortened poly(A) tracts, indicating that poly(A)+ RNA with shortened poly(A) tracts can be transported out of the nucleus. An experiment was designed to test the effect of 3'-deoxyadenosine triphosphate on the release of poly(A)+ RNA at drug concentrations which caused 33 or 64% inhibition of RNA release. The release of poly(A)+ RNA and poly(A)- RNA (not polyadenylated) was equally inhibited by the drug. Thus, although 3'-deoxyadenosine triphosphate does inhibit release of RNA from the nucleus, it would appear that the drug does so through a mechanism independent of the inhibition of polyadenylation. The process that is inhibited must be one that is common to both poly(A)+ and poly(A)- RNA. The possibility that 3'-deoxyadenosine triphosphate inhibits a reaction at the nuclear membrane or nuclear pore complex is considered.     

Multiple oligo(A) tracts associated with inactive sea urchin maternal mRNA sequences

Maternal RNA of sea urchin eggs and embryos was analyzed for short poly(A) sequences by digesting hybrids formed between [³H]poly(U) and poly(A) with RNase at 4°C. When the undigested [³H]poly(U) is precipitated with CTAB, all (A)_n tracts longer than 6 nucleotides are detected. This assay revealed a poly(A) content severalfold higher than is obtained with a similar assay using RNase at higher temperatures. On polyacrylamide gel electrophoresis, most of the previously undetected (A)_n tracts ran as a peak of oligo(A) of less than 20 nucleotides which accumulated at the dye front. The oligo(A) sequences were resolved into a single peak of (A)₁₀ when sized on Sephadex G100. These (A)₁₀ sequences were associated with large mRNA-sized molecules of about 3000 nucloetides average length which comprised 0.5 to 2% of the total maternal RNA. However, the (A)₁₀ sequences were not in mRNA molecules containing 3′-terminal poly(A) of 50–120 nucleotides nor did they remain in RNA that entered polysomes upon fertilization. However, hybridization studies showed that all sequences represented in the maternal poly(A)-containing RNA appeared to be present in the RNA molecules containing only (A)₁₀ sequences. The results suggest that the (A)₁₀-containing RNA might be incompletely processed mRNA precursor-like molecules.     

Mitochondrial poly(A) RNA synthesis during early sea urchin development

The synthesis of mitochondrial messenger RNA during early sea urchin development was examined. Oligo(dT) chromatography and electrophoresis on aqueous or formamide gels of mitochondrial RNA from pulse-labeled embryos showed the presence of eight distinct poly(A)-containing RNA species, ranging in size from 9 to 22 S. Nuclease digestion of these RNAs revealed poly(A) sequences of 4 S size. Using sea urchin anucleate fragments, we were able to demonstrate that all eight messenger RNAs are transcribed from mitochondrial DNA, rather than being transcribed from nuclear DNA and imported into the mitochondria.There was no change in the electrophoretic profile of the eight poly(A) RNAs when embryos were pulsed with [³H]uridine at various times after fertilization. Neither was there any change in the incorporation of [³H]uridine into these species or in the percentage of total newly synthesized mitochondrial RNA that contains poly(A) sequences as development progresses. Even though these RNAs appear to be transcribed at a constant rate throughout early development, they were not detected in mitochondrial polysomes until 18 hr after fertilization.     

The degradation of ribonucleic acids injected into Xenopus laevis oocytes

Different radioactive RNAs were injected into Xenopus laevis oocytes, and their degradation followed with time. Deproteinized ribosomal RNAs and synthetic polynucleotides, with the exception of polyadenylic acid, were degraded rapidly with apparent first order kinetics and half-lives ranging from 1–6 hr. Transfer RNA, poly(A), and ribosomal RNA injected as whole ribosomal particles were quite stable during the period studied (20 hr). Messenger RNAs from Dictyostelium discoideum and Vesicular Stomatitis Virus, which have poly(A) sequences at their 3′ terminus, presented biphasic degradation kinetics. Approximately 60% of these RNAs was degraded in the first 6 hr, whereas the remaining 30–40% was stable for at least 22 hr. Analysis of the stable material by sucrose gradients showed that it had the same sedimentation pattern as the original material, except that it contained, in addition, free poly(A) sequences sedimenting somewhat smaller than 4S. Puromycin treatment of the cells injected with Dictyostelium mRNAs reduced the percentage of stable RNA to 10%, approximately the poly(A) content of these RNAs. Similar treatment with emetine, which also inhibited cellular protein synthesis, did not affect the stable mRNA fraction.     

Characterization of polyadenylated RNA in a protein-producing bacterium, Bacillus brevis 47.

Up to about 50% of the total radioactivity in pulse-labeled RNA in Bacillus brevis 47-5, a high-protein-producing bacterium, was found in the polyadenylated fraction [termed poly(A)-RNA] isolated by adsorption to oligodeoxythymidylic acid-cellulose. Labeled RNA was bound to the cellulose regardless of whether the radioactive precursor was [3H]adenosine or [3H]uridine, showing that the adsorbed material was poly(A)-RNA rather than free poly(A). Poly(A) tracts, isolated after digestion of pulse-labeled RNA with pancreatic and T1 RNases, were homogeneous, with a length of about 95 nucleotides. Susceptibility of the isolated poly(A) tracts to degradation by snake venom phosphodiesterase and polynucleotide phosphorylase indicated that the poly(A) sequences were located directly at the 3'-terminal of the RNA molecules. Comparison of the poly(A)-RNA content in high-protein-producing and nonprotein-producing cells of B. brevis 47 showed much higher levels in the former. Electrophoretic analysis in both denaturing and denaturing polyacrylamide gels of the poly(A)-RNAs showed a heterogeneous population of molecules ranging in size from 23S to 4S. Comparison of the molecular-weight distribution patterns revealed that a significantly greater amount of high-molecular-weight poly(A)-RNA (comigrating with 23S RNA) was present under conditions in which extracellular protein production was high. The possibility that a substantial fraction of the poly(A)-RNA might be involved in the synthesis of extracellular proteins in B. brevis 47 is discussed.     

Isolation and translation in vitro of poly(A)+RNA from the free-living nematode Panagrellus silusiae.

Polysomal RNA was isolated from the free-living nematode Panagrellus silusiae. Passage of this RNA through a cellulose column resulted in the fractionation of the input RNA into poly(A)-RNA (ca. 97.5% of the total) and poly(A)+ RNA (ca. 2.5% of the total). RNase digestion, followed by polyacrylamide gel electrophoresis, revealed that the poly(A)+ RNA contained poly(A) tracts that ranged from 75 to 104 nucleotides in length with a mean value of about 90 residues. There was no evidence of poly(A) sequences in the poly(A)- RNA fraction. Poly(A)+ RNA gave a 25- to 50-fold stimulation (over background) of amino acid incorporation in the wheat germ cell-free protein-synthesizing system. At least 26 proteins were evident after electrophoresis in cylindrical sodium dodecyl sulfate-polyacrylamide gels. Poly(A)-RNA was capable of stimulating protein synthesis in vitro with about five discrete proteins being produced. In summary, the properties of mRNA from a simple organism such as P. silusiae are very similar to those of more complex eukaryotes.     

Biosynthesis of squalene and sterols by rat aorta

The synthesis of nonsaponifiable compounds from radioactive mevalonate by segments of adult rat aorta was studied in vitro. The labeled products consisted largely of substances with the chromatographic and chemical behavior of squalene, lanosterol, lathosterol, and cholesterol. Even after 3 or 4 hr of incubation, the incorporation of mevalonate into squalene was higher than its incorporation into C(27) sterols; cholesterol contained less than 20% of the radioactivity in the total sterols. Lanosterol was the most highly labeled sterol. The level of radioactivity in lathosterol was comparable to the level in cholesterol. Small amounts of radioactivity were found in other sterols. Material with the same mobility on TLC as 7-dehydrocholesterol had less radioactivity than cholesterol, but more than sterols with the mobility of desmosterol. The results of measurements made after short periods of incubation showed that squalene and lanosterol became labeled before the other nonsaponifiable compounds.     

Synthesis of angiotensinogen by isolated rat liver cells and its regulation in comparison to serum albumin

Angiotensinogen (renin substrate) and albumin are synthesized by isolated hepatocytes almost linearly for 5 hr. The incorporation of radioactive leucine into total protein proceeded linearly for 3 hr. Without addition of amino acids to the incubation medium the synthesis of both proteins was still linear but fell off to 40% compared to the synthesis rate obtained by incubation with amino acids in serum concentrations. Higher amino acid concentrations could not further stimulate the synthesis. Addition or withdrawal of tryptophan had no effect on the synthesis rate of both proteins. After 5 hr incubation hydrocortisone had stimulated the incorporation of radioactive leucine into total protein by 13%, the albumin synthesis by 43%, and the angiotensinogen synthesis by 142%.     

DNA sequence organization and transcription of the chicken genome

A new approach has been used to examine DNA sequence organization in the chicken genome. The interspersion pattern was determined by studying the fraction of labelled DNA fragments of different lengths that hybridized to an excess of short chicken repeated DNA sequences. The results indicate that chicken DNA has a pattern of sequence organization quite different than the standard ‘Xenopus’ or ‘Drosophila’ patterns. Two classes of unique sequences are found. One, 34% of the genome, consists of unique sequences approx. 4 kb long interspersed with repeated sequences. The second, non-interspersed fraction, 38% of the genome, consists of unique sequences found in long tracts, a minimum of approx. 22 kb in length. In an attempt to determine whether a relationship exists between DNA sequence organization and the distribution of structural genes we have isolated chicken DNA sequences belonging to different interspersion classes and tested each for the presence of structural genes by hybridization to excess poly(A)⁺ mRNA. Sequences complementary to poly(A)⁺ mRNA can be found with approximately the same frequency in both the non-interspersed fraction of the genome and a repeat-contiguous fraction enriched for interspersed sequences.     

DNA and RNA Syntheses by Intraerythrocytic Stages of Plasmodium knowlesi*

Incorporation of the nucleic acid precursors, orotic acid, adenosine, thymidine, and uridine, was studied in various stages of intraerythrocytic Plasmodium knowlesi from infected rhesus monkeys. Incubation of the parasitized erythrocytes with the precursors was for 3 hr periods using a plasma-free culture medium. The samples containing primarily rings, early trophozoites, or late trophozoites incorporated orotic acid, adenosine, and uridine into RNA; however, these stages exhibited negligible or very low levels of incorporation of any of the precursors into DNA. The sample containing late trophozoite and schizont stages incorporated orotic acid, adenosine, and uridine into RNA, and orotic acid, adenosine, and very low levels of thymidine into DNA. These results indicate that DNA synthesis (the S phase of the cell cycle) occurs very close to the time of nuclear division, and that either the G1 or G2 phase is very short in P. knowlesi. It was also observed that adenosine and orotic acid, 2 precursors which are incorporated into both DNA and RNA, are utilized differently by the intraerythrocytic parasites. Incorporation of orotic acid into RNA and DNA and adenosine incorporation into DNA were continuous for the entire incubation period, whereas incorporation of adenosine into RNA was very low during the last 2 hr of each period. It was further demonstrated that the parasites utilized exogenous uridine for synthesis of RNA, and that the older parasite stages incorporated thymidine into DNA.     

Synthesis of ribonucleic acid in normal bone in vitro

1. The incorporation of [2-(14)C]uridine into nucleic acids of bone cells was studied in rat and pig trabecular-bone fragments surviving in vitro. 2. The rapid uptake of uridine into trichloroacetic acid-soluble material, and its subsequent incorporation into a crude nucleic acid fraction of bone or purified RNA extracted from isolated bone cells, was proportional to uridine concentration in the incubation medium over a range 0.5-20.0mum. 3. During continued exposure to radioactive uridine, bulk RNA became labelled in a curvilinear fashion. Radioactivity rapidly entered nuclear RNA, which approached its maximum specific activity by 2hr. of incubation; cytoplasmic RNA, and particularly microsomal RNA, was more slowly labelled. The kinetics of labelling and rapid decline of the nuclear/microsomal specific activity ratio were consistent with a precursor-product relationship. 4. Bulk RNA preparations were resolved by zonal centrifugation in sucrose density gradients into components with approximate sedimentation coefficients 28s, 18s and 4s. 5. Rapidly labelled RNA, predominantly nuclear in location, demonstrated a polydisperse sedimentation pattern that did not conform to the major types of stable cellular RNA. Material of highest specific activity, sedimenting in the 4-18s region and insoluble in 10% (w/v) sodium chloride, rapidly achieved its maximum activity during continued exposure to radioactive precursor and decayed equally rapidly during ;chase' incubation, exhibiting an average half-life of 4.3hr. 6. Ribosomal 28s and 18s RNA were of lower specific activity, which increased linearly for at least 6hr. in the continued presence of radioactive uridine. There was persistent but variable incorporation into ribosomal RNA during ;chase' incubation despite rapid decline in total radioactivity of the acid-soluble pool containing RNA precursors.     

Polyadenylic acid sequences in the RNA of Hyphomicrobium

Heterogeneous RNA containing polyadenylic acid [poly(A)] sequence has been isolated from Hyphomicrobium by affinity chromatography on oligothymidylic acid cellulose and polyuridylic acid Sepharose columns. About 0.1 to 0.3% of [3H]adenine-labeled RNA over a 60-min period is associated with poly(A) sequences. This percentage decreases to about 0.03 in a 20-h labeling period. The poly(A) tracts recovered after digestion with ribonuclease A and T1 are composed of greater than 95% adenine residues and are up to 200 nucleotides in length with a predominant range of 15 to 40 nucleotides. Adenosine and AMP are present in the ratio of 1:36 in alkaline digests of Hyphomicrobium poly(A) tracts. This is compatible with nucleotide lengths determined on acrylamide gels and location at the 3'-OH terminus of the RNA molecule.     

Messenger RNA in the cytoskeletal framework: analysis by in situ hybridization

The possibility of an association of mRNA with the cytoskeletal framework (CF) of ascidian (Styela plicata) follicle cells was examined in this study. The approach was to extract the follicle cells with Triton X-100 and determine whether mRNA persisted in the insoluble residue by two methods, in situ hybridization with poly(U) and actin DNA probes and the incorporation of radioactive isotopes into RNA. Triton X-100 extraction of follicle cells yielded a filamentous CF containing approximately 70% of the total poly (A) but only 9% of the total lipid, 23% of the total protein, and 28% of the total RNA. In situ hybridization with a poly (U) probe indicated that approximately 70% of the poly (A) was associated with the CF. In situ hybridization with a cloned actin DNA probe indicated that approximately 60% of the actin mRNA was associated with the CF. Autoradiography of detergent- extracted follicle cells, which had been labeled with [3H]uridine or [3H]adenosine, indicated that greater than 90% of the newly synthesized poly (A)+RNA was preserved in the CF. Thus more newly synthesized mRNA than steady-state mRNA may be present in the Triton X-100 insoluble fraction. It is concluded that a significant proportion of the mRNA complement of ascidian follicle cells is associated with the CF.