Analysis of the membrane-associated poly(A)+RNA in the cytoplasm of dormant Artemia cysts by DNA excess hybridization. Evidence for a nuclear origin

Encysted embryos of Artemia contain latent mRNA, to a large extent associated with a fraction of cytoplasmic membranes. The membranes, purified by EDTA treatment and banding in a sucrose gradient at 1.17 g/cm3, include endoplasmic vesicles and mitochondria. The origin of the membrane-associated poly(A)+RNA was therefore investigated. In gel electrophoresis poly(A)+RNA from the purified membranes of dormant cysts forms two distinct bands at approx. 3 . 10(5) and 5 . 10(5) Da. Later during development the lighter component decreases. Nuclei from dormant cysts are devoid of poly(A)+RNA, while nuclei from developing embryos (50% emergence) contain a predominant poly(A)+RNA component of approx. 5 . 10(5) Da. 125I-labelled preparations of nuclear DNA and of nuclear and membrane-associated poly(A)+RNA were used in reassociation and hybridization experiments with excess nuclear DNA. Poly(A)+RNA from the membranes of dormant cysts hybridized to nuclear DNA to the same extent as the nuclear poly(A)+RNA from developing embryos. The hybridization of labelled, nuclear poly(A)+RNA to nuclear DNA was strongly inhibited by unlabelled membrane RNA from either dormant cysts or developing embryos. It is concluded that the stored, membrane-associated poly(A)+RNA in dormant cysts is essentially of nuclear origin. The 5 . 10(5)-Da component is largely homologous with the corresponding component of nuclear poly(A)+RNA at later stages.     

The expression of a gene for eukaryotic elongation factor Tu in Artemia during development. Translation of poly(A)+ RNA and the use of a synthetic oligonucleotide to detect the presence of eukaryotic elongation factor Tu-specific mRNA

Total in vivo proteins from Artemia embryos at different developmental stages were examined by two-dimensional gel electrophoresis. A variety of peptides change during development, with one of them, the eukaryotic elongation factor Tu (eEF-Tu), presenting a dramatic increase from dormant embryos to nauplii. When poly(A)+ RNA is translated in vitro, the same relative increase is seen for eEF-Tu during development. Based on the amino acid sequence for Artemia eEF-Tu (Amons, R., Pluijms, W., Roobol, K., and M?ller, W. (1983) FEBS Lett. 153, 37-42), a synthetic oligodeoxynucleotide was prepared and used to prime the synthesis of cDNA with poly(A)+ RNA from 12-h developing embryos as template. Direct sequence analysis of the 900-base primary cDNA product shows it to be specific for the 5' end of Artemia eEF-Tu mRNA. Hybridization of a "Northern" blot of denatured (poly(A)+ RNA from different developmental stages with this cDNA reveals a major band migrating at about 1800 bases, which increase in intensity as development proceeds, paralleling the increase in eEF-Tu seen by in vitro translation. When poly(A)+ RNA is separated on a nondenaturing gel, blotted to poly(U) paper, and hybridized with the eEF-Tu cDNA, a single band is observed migrating faster than 18 S. Elution and in vitro translation of this band results in a major product migrating with eEF-Tu in a dodecyl sulfate-polyacrylamide gel and which is precipitable with eEF-Tu-specific antibodies.     

Gene activity during germination of spores of the fern, Onoclea sensibilis. Cell-free translation analysis of mRNA of spores and the effect of alpha-amanitin on spore germination

Poly(A)-RNA fractions of dormant, dark-imbibed (non-germinating) and photoinduced (germinating) spores of Onoclea sensibilis were poor templates in the rabbit reticulocyte lysate protein synthesizing system, but the translational efficiency of poly(A)+RNA was considerably higher than that of unfractionated RNA. Poly(A)+RNA isolated from photoinduced spores had a consistently higher translational efficiency than poly(A)+RNA from dark-imbibed spores. Analysis of the translation products by one-dimensional polyacrylamide gel electrophoresis showed no qualitative differences in the mRNA populations of dormant, dark-imbibed, and photoinduced spores. However, poly(A)+RNA from dark-imbibed spores appeared to encode in vitro fewer detectable polypeptides at a reduced intensity than photoinduced spores. A DNA clone encoding the large subunit of maize ribulose bisphosphate carboxylase hybridized at strong to moderate intensity to RNA isolated from dark-imbibed spores, indicating the absence of mRNA degradation. Although alpha-amanitin did not inhibit the germination of spores, the drug prevented the elongation of the rhizoid and protonemal initial with a concomitant effect on the synthesis of poly(A)+RNA. These results are consistent with the view that some form of translational control involving stored mRNA operates during dark-imbibition and photoinduced germination of spores.     

Characterization of a poly(A)+RNA-containing structural component directly associated with cytoplasmic membranes in dormant Artemia cysts

Poly(A)+RNA-containing material was extracted from the purified cytoplasmic membranes of dormant Artemia cysts by treatment with mild detergents. Sedimentation analysis of the extracts showed a predominant poly(A)-containing fraction at 40 S, associated with about 6% of the extracted proteins. Only limited amounts of poly(A)-containing material were found in the heavier fractions. Poly(A)+RNA extracted from the 40-S fraction sedimented around 14 S. The poly(A)-containing 40-S structures could be purified by treatment with non-ionic or zwitterionic detergents followed by resedimentation in sucrose gradients in the presence or absence of detergent. When the 40-S fraction was analyzed by isopycnic centrifugation in Cs2SO4 gradients, the main part of the poly(A)-containing material banded at a density of 1.27 g/ml. Electron-microscopic examination of this fraction revealed circular or slightly bullet-shaped profiles measuring 17-26 nm. When the 40-S fraction had been submitted to mild RNAase treatment prior to density gradient centrifugation, the material was displaced towards lower density and became less distinct. Purified 40-S particles showed a complex protein pattern not very similar to that of polyribosomal poly(A)+RNA-containing particles from developing embryos, but with components in common with unfractionated membranes. The particles also contained some lipids. The experiments indicate that a major part of the membrane-bound, latent poly(A)+RNA in dormant Artemia cysts occurs in the form of relatively uniform, detergent- and Cs2SO4-resistant structures, independent of ribosomes, but intimately associated with membrane components.     

Developmental changes in poly(A) polymerase activity in Artemia

The levels of poly(A) polymerase activity have been determined during Artemia early development. Poly(A) polymerase activity increases steadily during postgastrular embryonic development reaching a maximum shortly after hatching. The rise of poly(A) polymerase is concomitant with an increase in poly(A) content and with a change in the subcellular distribution of the enzyme activity, the major increase corresponding to the nuclear fraction. Only one isoenzyme of poly(A) polymerase has been identified in Artemia embryos and nauplii despite changes in enzyme levels and subcellular changes during early development. Poly(A) polymerase is not associated with the cytoplasmic poly(A)-containing ribonucleoprotein particles stored in Artemia dormant embryos.     

Changes in hepatic RNA, poly(A)+RNA, and poly(A)-RNA during the acute phase response to inflammation

The steady state changes in total rat hepatic cytoplasmic RNA, poly(A)+ RNA and poly(A)-RNA were assessed in response to turpentine induced inflammation. From 18 to 24 h after injury, cytoplasmic RNA doubled, while poly(A)+ RNA peaked at 24 h, 3.5 times over control animals. Cell-free translation showed significant increases in messenger RNA levels beginning at 18 h. Gel electrophoresis of translation products revealed significant increases in several polypeptides and a decrease in others. Poly(A)-RNA from control and injured rats translated to an insignificant level and the electrophoretic gel patterns of their proteins were similar. Furthermore, no change had occurred in the 3' poly(A)-sequences during the course of inflammation.     

The metabolic behaviour of nuclear and cytoplasmic non-polyadenylated RNAs with an affinity for poly(adenylic acid) from Friend murine leukaemia cells

Using poly(A)-Sepharose and poly(U)-Sepharose affinity chromatography, various classes of nuclear RNA can be distinguished in Friend leukaemia cells. One of these contains a poly(A) tract (poly(A)+-RNA) and another lacks a poly(A) tract but has an affinity for poly(A)-Sepharose (poly(A)-u+-RNA). The stability of these two particular nuclear RNA classes was examined by using a 'pulse-chase' technique involving D-glucosamine treatment. Nuclear poly(A)-u+-RNA was found to decay as a single component with a half-life of about 12 min. In contrast, nuclear poly(A)+-RNA appears to consist of at least two distinct metabolic components with half-lives of about 22 min and 120 min. Furthermore, poly(A)-u+-RNA is transported from the nuclei much more rapidly than the poly(A)+-RNA. The 'pulse-chase' approach also allowed a quantitative estimate to be made of the conversion of nuclear poly(A)+-RNA and poly(A)-u+-RNA to cytoplasmic poly(A)-RNA and poly(A)-u+-RNA.     

Structure, translation, and metabolism of the cytoplasmic copia ribonucleic acid of Drosophila melanogaster

We have characterized the copia RNA in the cytoplasm of cultured Drosophila cells. Copia RNA was detected and purified by hybridization to DNA of the plasmid cDm 1142, which contains the copia sequence. A large fraction (2.2%) of the total cytoplasmic poly(A)+ RNA was found to be copia RNA. Cytoplasmic copia RNA displays all the characteristics expected for a messenger RNA. It possesses a poly(A) tract identical in length with that of total poly(A)+ cytoplasmic RNA. It is associated with polysomes and can be released from this association by treatment with EDTA. When purified copia RNA is added to an mRNA-dependent rabbit reticulocyte lysate, three polypeptides of 51000, 33000, and 21000 daltons are seen. We have not determined if these are different polypeptides or if the two smaller polypeptides are fragments of the 51000-dalton polypeptide. The half-life of copia cytoplasmic RNA was determined in pulse--chase experiments to be 9.5 h; this is 1.6 times longer than the half-life of the intermediate decay class of total poly(A)+ cytoplasmic RNA. These properties provide strong evidence that copia RNA functions in vivo as a messenger RNA.     

Stored Messenger Ribonucleoprotein Particles in Differentiated Sclerotia of Physarum polycephalum

Starvation induces vegetative microplasmodia of Physarum polycephalum to differentiate into translationally-dormant sclerotia. The existence and the biochemical nature of stored mRNA in sclerotia is examined in this report. The sclerotia contain about 50% of the poly(A)-containing RNA [poly(A)+RNA] complement of microplasmodia as determined by [³H]-poly(U) hybridization. The sclerotial poly(A)+RNA sequences are associated with proteins in a ribonucleoprotein complex [poly(A)+mRNP] which sediments more slowly than the polysomes. Sclerotial poly(A)+RNP sediments more rapidly than poly(A)+RNP derived from the polysomes of microplasmodia despite the occurrence of poly(A)+RNA molecules of a similar size in both particles suggesting the existence of differences in protein composition. Isolation of poly(A)+RNP by oligo (dT)-cellulose chromatography and the analysis of its associated proteins by polyacrylamide gel electrophoresis show that sclerotial poly(A)+RNP contains at least 14 major polypeptides, 11 of which are different in electrophoretic mobility from the polypeptides found in polysomal poly(A)+RNP. Three of the sclerotial poly(A)+RNP polypeptides are associated with the poly(A) sequence (18, 46, and 52 × 10³ mol. wt. components), while the remaining eight are presumably bound to non-poly(A) portions of the poly(A)+RNA. Although distinct from polysomal poly(A)+RNP, the sclerotial poly(A)+RNP is similar in sedimentation behavior and protein composition (with two exceptions) to the microplasmodial free cytoplasmic poly(A)+RNP. The results suggest that dormant sclerotia store mRNA sequences in association with a distinct set of proteins and that these proteins are similar to those associated with the free cytoplasmic poly(A)+RNP of vegetative plasmodia.     

Cellular titers and subcellular distributions of abundant polyadenylate-containing ribonucleic acid species during early development in the frog Xenopus laevis.

The distribution of cytoplasmic messenger ribonucleic acids (RNAs) in translationally active polysomes and inactive ribonucleoprotein particles changes during early development. Cellular levels and subcellular distributions have been determined for most messenger RNAs, but little is known about how individual sequences change. In this study, we used hybridization techniques with cloned sequences to measure the titers of 23 mitochondrial and non-mitochondrial polyadenylate-containing [poly(A)+]RNA species during early development in the frog Xenopus laevis. These RNA species were some of the most abundant cellular poly(A)+ RNA species in early embryos. The concentrations of most of the non-mitochondrial (cytoplasmic) RNAs remained constant in embryos during the first 10 h of development, although the concentrations of a few species increased. During neurulation, we detected several new poly(A)+ RNA sequences in polysomes, and with one possible exception the accumulation of these sequences was largely the result of new synthesis or de novo polyadenylation and not due to the recruitment of nonpolysomal (free ribonucleoprotein) poly(A)+ RNA. We measured the subcellular distributions of these RNA species in polysomes and free ribonucleoproteins during early development. In gastrulae, non-mitochondrial RNAs were distributed differentially between the two cell fractions; some RNA species were represented more in free ribonucleoproteins, and others were represented less. By the neurula stage this differential distribution in polysomes and free ribonucleoproteins was less pronounced, and we found species almost entirely in polysomes. Some poly(A)+ RNA species transcribed from the mitochondrial genome were localized within the mitochondria and were mapped to discrete fragments of the mitochondrial genome. Much of this poly(A)+ RNA was transcribed from the ribosomal locus. Nonribosomal mitochondrial poly(A)+ RNA species became enriched in polysome-like structures after fertilization, with time courses similar to the time course of mobilization of cytoplasmic poly(A)+ RNA.     

The preparation and characterization of locust vitellogenin messenger RNA and the synthesis of its complementary DNA.

Poly(A)+ (polyadenylated) RNA was isolated from vitellogenic female-locus fat-body by LiCl/urea extraction and poly(U)-Sepharose 4B affinity chromatography. Agarose-gel electrophoresis of this poly(A)+ RNA under denaturing conditions shows the presence of a high-molecular-weight species (greater than 31 S, 7100 nucleotides) as the major species, which is absent from the RNA prepared from male-locust fat-body. Inclusion of this poly(A)+ RNA in a mRNA-dependent reticulocyte-lysate system directs the synthesis of polypeptides that could be immunoprecipitated with monospecific antibodies against locust egg vitellin. DNA complementary (cDNA) to the poly(A)+ RNA was synthesized, and back-hybridization of the cDNA to its template reveals a major abundant species comprising about 45% of the total poly(A)+ RNA hybridizing with R0t 1/2 of 2 x 10(-2) mol . litre-1 . s. Abundant cDNA isolated from the total cDNA hybridizes to poly(A)+ RNA with a R0t 1/2 of 9 x 10(-3) mol . litre-1 . s. There are 9.1 x 10(3) copies of vitellogenin mRNA per cell of vitellogenic female-locust fat-body, comprising 55% of the poly(A)+ RNA and equivalent to 0.7% of total cellular RNA.     

Analysis of the membrane-associated poly(A)+RNA in the cytoplasm of dormant Artemia cysts by DNA excess hybridization: Evidence for a nuclear origin

Encysted embryos of Artemia contain latent mRNA, to a large extent associated with a fraction of cytoplasmic membranes. The membranes, purified by EDTA treatment and banding in a sucrose gradient at 1.17 g/cm³, include endoplasmic vesicles and mitochondria. The origin of the membrane-associated poly(A)⁺RNA was therefore investigated. In gel electrophoresis poly(A)⁺RNA from the purified membranes of dormant cysts forms two distinct bands at approx. 3·10⁵ and 5·10⁵ Da. Later during development the lighter component decreases. Nuclei from dormant cysts are devoid of poly(A)⁺RNA, while nuclei from developing embryos (50% emergence) contain a predominant poly(A)⁺RNA component of approx. 5·10⁵ Da. ¹²⁵I-labelled preparations of nuclear DNA and of nuclear and membrane-associated poly(A)⁺RNA were used in reassociation and hybridization experiments with excess nuclear DNA. Poly(A)⁺RNA from the membranes of dormant cysts hybridized to nuclear DNA to the same extent as the nuclear poly(A)⁺RNA from developing embryos. The hybridization of labelled, nuclear poly(A)⁺RNA to nuclear DNA was strongly inhibited by unlabelled membrane RNA from either dormant cysts or developing embryos. It is concluded that the stored, membrane-associated poly(A)⁺RNA in dormant cysts is essentially of nuclear origin. The 5·10⁵-Da component is largely homologous with the corresponding component of nuclear poly(A)⁺RNA at later stages.     

Preformed and newly synthesized messenger RNA in germinating wheat embryos

In 6 h germinated wheat (Triticum aestivum L. cv. Cama) embryos, more than half of the messenger RNAs are actively involved in translation. Neither preformed nor newly synthesized poly A⁺-RNA is translated preferentially. Germination in the presence of cordycepin showed that the half-life of the templates is about 2 h and that the newly synthesized messengers are essential to support protein synthesis in the embryo from the first hours of germination. Most of the messenger RNAs in 6 h germinated embryos are newly synthesized. The polypeptides coded for by either the endogenous messenger ribonucleoproteins or purified poly A⁺-RNA from both dry and germinated embryos are qualitatively identical; minor quantitative differences can however be observed.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate - TCA trichloroacetic acid - mRNP messenger ribonucleoprotein - poly A⁺-RNA polyadenylic acid containing RNA - PB polysome buffer - GM germination medium     

Size and turnover of polyadenylic acid-containing ribonucleic acids in a fragile mutant of Saccharomyces cerevisiae.

Ribonucleic acid-containing polyadenylic acid [poly(A)+-RNA] was studied in lysates from an osmotic-sensitive mutant of Saccharomyces cerevisiae characterized by low nuclease activity. The poly(A)+-RNA fraction, analyzed by electrophoresis in polyacrylamide-formamide gels, constitutes a heterogeneous population of molecules, with molecular weights ranging from 0.2 X 10(6) to 3 X 10(6) and having an average of 1.2 X 10(6). The turnover rate of poly(A)+-RNA was determined by the decay of radioactivity after a cold uracil chase, and the observed half-life of 21 min corresponds to about 10% of the cell doubling time. Poly(A)+-RNA was analyzed by gel electrophoresis under denaturing and non-denaturing conditions. A correlation was established between the apparent secondary structure and the turnover rate of poly(A)+-RNA species.     

Template activity for histones of poly(A)-minus RNA fraction from different developmental stages of Artemia salina embryos

Template activity for histones of RNA fractions derived from Artemia salina embryos at different developmental stages were measured in a Krebs ascites cell-free system. Appreciable amounts of acid-soluble polypeptides comigrating with Hela cell histone markers on acrylamide gel electrophoresis were detected only when RNA fractions from nauplii were used. Tryptic peptide analysis by high voltage electrophoresis of the translational products had a pattern qualitatively similar to that of in vivo labeled histone markers from Hela.