Polyadenylic acid-containing RNA and its polyadenylic acid sequences newly synthesized in isolated embryonic cells of Xenopus laevis.

Newly synthesized polyriboadenylic acid [poly(A)]-containing RNA and its poly(A) sequences were isolated and characterized in Xenopus embryonic cells. Upon sedimentation analysis, the poly(A)-containing RNA labeled for 30 min showed a very heterogeneous size distribution ranging from 9 to >40 S. After 5 hr of labeling, the profile became much less heterogeneous and the main component was distributed in the 9–28 S region. The average molecular weight of 6.5–7.0 × 10⁵ daltons was calculated for the 5-hr labeled RNA. This poly(A)-containing RNA, comprising about 10% of the total labeled RNA, was metabolically stable and accumulated linearly for 5 hr. Gel electrophoresis of the RNA revealed the presence of little or no free poly(A) sequences. Most of the poly(A) sequences, which were isolated from 30-min labeled poly(A)-containing RNA migrated as a single discrete component approximately 150 nucleotides long. In contrast, they were slightly smaller (130 nucleotides long) and more heterogeneous, when obtained from the poly(A)-containing RNA labeled for 5 hr. From these results, it may be likely that the embryonic poly(A)-containing RNA is similar in size to the steady-state population of the poly(A)-containing RNA reported to occur in vitellogenic oocytes and cultured kidney cells of the same species.     

Relative amounts of newly synthesized poly(A)+ and poly(A)- messenger RNA during development of Xenopus laevis

The relative amounts of newly synthesized poly(A)⁺ and poly(A)^? mRNA have been determined in developing embryos of the frog Xenopus laevis. Polysomal RNA was isolated and fractionated into poly(A)⁺ and poly(A)^? RNA fractions with oligo(dT)-cellulose. In normal embryos the newly synthesized polysomal poly(A)⁺ RNA has a heterodisperse size distribution as expected of mRNA. The labeled poly(A)^? RNA of polysomes is composed mainly of rRNA and 4S RNA. The amount of poly(A)^? mRNA in this fraction cannot be quantitated because it represents a very small proportion of the labeled poly(A)^? RNA. By using the anucleolate mutants of Xenopus which do not synthesize rRNA, it is possible to estimate the percentage of mRNA which contains poly(A) and lacks poly(A). All labeled polysomal RNA larger than 4S RNA which does not bind to oligo(dT)-cellulose in the anucleolate mutants is considered presumptive poly(A)^? mRNA. The results indicate that about 80% of the mRNA lacks a poly(A) segment long enough to bind to oligo(dT). The poly(A)⁺ and poly(A)^? mRNA populations have a similar size distribution with a modal molecular weight of about 7 × 10⁵. The poly(A) segment of poly(A)⁺ mRNA is about 125 nucleotides long. Analysis of the poly(A)^? mRNA fraction has shown that it lacks poly(A)₁₂₅.     

Cytogenetic analysis of oocytes and early preimplantation embryos from XO mice.

The cytoplasm of early sea urchin embryos contains nonribosomal, high molecular weight RNA both associated with ribosomes in polysomes and free of ribosomes in particles termed free RNP. In a 1-hr labeling period, 50% of the newly synthesized RNA enters the pool of ribosome-free RNP particles during the cleavage stages, and this percentage decreases until less than 20% of the new RNA in the mesenchyme blastula stage is found in the free RNP. mRNA from both polysomes and free RNP contain poly(A)(+) and poly(A)(?) species. During the cleavage stages only 8–10% of the RNA from each fraction is polyadenylated; however, in the blastula, 40–50% of the nonhistone polysomal RNA is polyadenylated while only 22–30% of the free RNP RNA is polyadenylated. At any developmental stage, the poly(A)(+)RNA from the free RNA and polysomes have identical sedimentation profiles; this is also the case for the poly(A)(?)RNA except for the absence of the 9 S histone mRNA from the free RNP. Changes in poly(A)(+)RNA content and sedimentation profiles during development occur simultaneously in the free RNP and the polysomes. Kinetic studies of these two RNP populations as well as nuclear RNP show that the bulk of the free RNP are not unusually stable cytoplasmic components. The free RNP decay with a half-life of about 40 min while nuclear RNA and polysomal RNA display half-lives of about 12 and 65 min, respectively. Further, the rate of synthesis of the free RNP is not consistent with their being the only precursors for polysomes. Our estimates of the rates of synthesis for nuclear RNA, polysomes, and free RNP are, respectively, 1.1 × 10^?15, 2.2 × 10^?16, and 5.0 × 15^?17 g/min/nucleus. The data on free RNP is discussed in terms of translational regulation of protein synthesis in the developing sea urchin.     

Variations in the amount of polysomes in mature oocytes of Drosophila melanogaster.

Quantitative measurements of polysomes and ribosomes of Drosophila melanogaster egg chambers, mature oocytes, and embryos were done using sucrose gradient analysis. The amount of polysomes per egg chamber increases about 20 times from stage 5 to 13, and then remains constant up to the end of embryogenesis. The percentage of ribosomes in polysomes is fairly constant during oogenesis and embryogenesis (56 ± 7%). Depending on the fly population, the percentage of ribosomes in polysomes of mature oocytes varies from 10 to 70%. It is shown that the percentage of polysomes in mature oocytes decreases with the time of retention of the mature oocytes in the ovary. Twenty-four- to thirty-six-hour-old flies kept in optimal conditions retain their mature oocytes for 2–3 hr. These mature oocytes still contain 40–60% ribosomes in polysomes. Conditions are given which allow the obtainment of reproducibly high amounts of polysomes from mature oocytes of Drosophila.     

Changing rates of DNA and RNA synthesis in Drosophila embryos

Rates of DNA and RNA synthesis during Drosophila embryogenesis were measured by labeling octane-treated embryos with [¹⁴C]thymidine and [³H]uridine. Radioactivity incorporated per hour was converted to rates of synthesis using measurements of the pool-specific activity during the labeling periods. The rate of DNA synthesis during early embryogenesis increases to a maximum at 6 hr after oviposition and then decreases sharply. Measured rates of DNA synthesis were used to calculate that the total amount of DNA per embryo doubles every 18 min at blastoderm, every 70–80 min during gastrulation, and less than once every 7 hr at later stages. The rate of RNA accumulation per embryo increases continuously during the first 14 hr of embryogenesis. The rate of nuclear RNA synthesis per diploid amount of DNA, however, decreases fivefold between blastoderm and primary organogenesis. The cytoplasmic poly(A)⁺ RNA synthesized by blastoderm embryos associates rapidly with polysomes. The relatively high rate of synthesis of polysomal poly(A)⁺ RNA per nucleus at blastoderm allows the small number of nuclei present at blastoderm to make a significant quantitative contribution to the informational RNA active in the early embryo. At the end of blastoderm, approximately 14% of the mRNA being translated in the embryo has been synthesized after fertilization.     

Mobilization of newly synthesized RNAs into polysomes inXenopus laevis embryos

Summary The mobilization of newly synthesized 18S and 28S rRNAs, 4S RNA and poly(A)⁺ RNA into polysomes was studied in isolated cells ofXenopus laevis embryos between cleavage and neurula stages. Throughout these stages, 4S RNA and poly(A)⁺ RNA were mobilized immediately following their appearance in the cytoplasm. 18S rRNA however, stayed in the ribosomal subunit fraction for about 30 min until the 28S rRNA appeared, when the two rRNAs were mobilized together at an equimolar ratio. This mobilization, at a 1:1 molar ratio, appeared to be realized at initiation monome formation. Thus, the efficiency of the mobilization of two newly synthesized rRNAs, shortly after their arrival at the cytoplasm, differed considerably but difference disappeared once steady state was reached.The contribution of newly synthesized 18S and 28S rRNAs to polysomes remains small throughout early development. around 3% of newly synthesized 4S RNA is polysomal which is the same distribution observed for unlabeled 4S RNA. Less than 10% of the newly synthesized cytoplasmic poly(A)⁺ RNA was mobilized into polysomes during cleavage, but in later stages the proportion increased to around 20%–25%. These results show that newly synthesized RNAs are utilized for protein synthesis at characteristic rates soon after they are synthesized during early embryonic development. On the basis of the data presented here and elsewhere we discuss quantitative aspects of the utilization of newly synthesized and maternal RNAs during early embryogenesis.     

Poly(A)+ RNA metabolism during oogenesis in Xenopus laevis.

In this study, we have measured the synthesis and turnover of oligo(dT)cellulose-bound RNA [poly(A)⁺ RNA] in Xenopus laevis oocytes at the maximal lampbrush chromosome stage (stage 3) and at the completion of oocyte growth (stage 6). Oocytes at both stages are shown to be active in the synthesis of poly(A)⁺ RNA. In stage 6 oocytes, the mean rate of synthesis of stable poly(A)⁺ RNA is 15% the instantaneous rate of synthesis, while the mean half-life of the unstable component is 1.6 hr. In contrast, the instantaneous rate of synthesis in stage 3 oocytes is about one-third that seen in stage 6, and most of it is devoted to the production of unstable species with an average half-life of 5 hr. Studies on the nuclear versus the cytoplasmic distribution of the newly synthesized poly(A)⁺ RNA demonstrated that by the end of a 12-hr labeling period for stage 3 oocytes and a 24-hr labeling period for stage 6 oocytes, approximately half of the material was cytoplasmic. This cytoplasmic material had the same electrophoretic mobility as bulk poly(A)⁺ RNA. Similarly, as with bulk poly(A)⁺ RNA, little, if any, of the newly synthesized material was found to be polysomal. Also, poly(A) labeling studies indicated that the newly synthesized poly(A)⁺ RNA was associated with the synthesis of poly(A) of the same length as that appearing on bulk poly(A)⁺ RNA. Studies on the content of bulk oligo(dT)cellulose-bound RNA indicated that about 86 ng is present in both stage 3 and stage 6 oocytes. The continual synthesis of poly(A)⁺ RNA throughout oogenesis in the absence of its accumulation led to the conclusion that it must be turning over. These data are discussed in relation to the hypothesis that bulk levels of poly(A)⁺ RNA are maintained by continually changing rates of synthesis and degradation.     

Alterations in polyadenylated RNA during pollen maturation and germination

Developmental changes in poly(A)-bearing RNA in male tobacco gametophyte were examined by sedimentation analysis and by hybridization with³H-poly(U). The results indicate that the transition of microspore undergoing postmeiotic division to mature pollen is accompanied by characteristic changes in RNA and poly(A) content and the size of poly(A)⁺RNA. The volume of pollen grain increases about 2times, total RNA per grain from 34 to 230 pg and poly(A) from 22 to 450 fg, which together with the estimated increase in the number average size of poly(A)⁺RNA from 700 to 2 100 nucleotides suggests an approx. rise of RNA containing poly(A) from 0.3 to 2.7% of total RNA. Size distribution of the populations of polyadenylated RNAs shows progressive formation of species with a higher molecular mass and differentiation of the pollen-characteristic pattern with main sedimentation maxima close to 12S, 19S and 26S. This pattern remains almost unchanged during 8 h of pollen tube growth and is also found in polysomes formed at the beginning of germination. The amount of poly(A) decreases gradually after the onset of soaking at a rate of slightly more than 1 % per h within 24 h of pollen cultivation. As a whole, the results demonstrate that in the course of pollen maturation a specific population of polyadenylated mRNAs is formed which persists as stored mRNA in quiescent pollen and is used as template during-pollen tube formation.     

Characterization of poly(A+)RNA in free messenger ribonucleoprotein and polysomes of mouse Taper ascites cells

Cytoplasmic extracts of mouse Taper ascites cells were centrifuged on sucrose gradients to give 0–80 S, monosome, and polysome fractions. CsCl equilibrium density centrifugation of formaldehyde-fixed material from the 0–80 S fraction demonstrated that the messenger RNA in the 0–80 S fraction was in the form of free ribonucleoprotein. The size of the poly(A⁺)RNA and the size of the poly(A) segments of these molecules were shown to be very similar in both the free mRNP² and polysome fractions. The labeling kinetics of the free mRNP poly(A⁺)RNA was similar to that of the polysomal poly(A⁺)RNA.The free mRNP poly(A⁺)RNA efficiently stimulated protein synthesis in the wheat germ cell-free system, supporting the view that it was mRNA. Two-dimensional gel electrophoresis was used to analyze the proteins whose synthesis was directed by free mRNP and polysomal poly(A⁺)RNA. The free mRNP poly(A⁺)RNA directed the synthesis of a simpler set of abundant protein products than did the polysomal poly(A⁺)RNA. Most of the free mRNP abundant protein products were also present in the polysomal products, though obvious quantitative differences were evident, indicating that each individual mRNA had its own characteristic distribution between polysomes and the translationally inactive RNP form.     

Stored and polysomal ribosomes of mouse ova

RNP particles of ovulated mouse ova, labeled by exposure of growing oocytes to [³H]uridine, were displayed on sucrose gradients. Under standard salt conditions, radioactivity was observed coinciding with liver ribosomal subunits, monomers, and polysomes. The RNA from each region of the gradient was isolated and was found to contain the expected species of labeled 18S and/or 28S ribosomal RNA. Heterogeneous RNP particles were widely distributed in the gradient. From data on RNase sensitivity and resistance to dissociation in high salt, it was estimated that 20–25% of the total ribosomes were in polysomes. No difference in the distribution was observed when ribosomes were labeled in the early or late growth phase of the oocyte. The evidence suggested that the nonpolysomal subunits and monomers were unable to form a high salt-stable complex in the presence of poly(U) and factors for protein synthesis. Thus, the bulk of the ribosomes are inactive in protein synthesis in ovulated ova and are apparently stored for use in embryonic development.     

Modulation of poly(A)+ RNA levels in fungal-infected wheat embryos through the selective inactivation of RNA polymerase II and poly(A) polymerase

Infection of germinating wheat embryos by a fungal pathogen (Drechslera sorokiana) drastically lowered (70–73%) the relative abundance of poly(A)⁺ RNA. This was paralleled by a significant loss in the activities of RNA polymerase II (60–70%) and poly(A) polymerase (80–85%) enzymes. The inhibition of RNA polymerase II (60–65%) and poly(A) polymerase (70–85%) activities was also witnessed by the in vitro addition of the fungal extract to the enzyme preparations isolated from healthy embryos. The fungal extract showed negligible phosphatase and nuclease activities. This ruled out the possibility of rapid degradation of the labelled substrate [³H]ATP, primer RNA, or even the labelled reaction products under our assay conditions. The inhibitory effect of the fungal extract could be alleviated by fractionating the treated enzyme preparation by phosphocellulose chromatography. This indicated that the fungal extract was directly responsible for the inactivation of the polymerases in a reversible manner. The inhibitory function of the fungal extract was destroyed by treatment with pronase, but not with RNAase A and RNAase Ti. Poly(A) ‘tails’ were enzymatically excised from ³²P-labelled poly(A)⁺ RNA and fractionated on acrylamide gels for autoradiographic analysis. The lengths of the ³²P-labelled poly(A) ‘tails’ in control and infected embryos turned out to be identical (64 nucleotides). Our results suggest that the relative abundance of poly(A)⁺ RNA is diminished in fungal-infected wheat embryos through the selective inactivation of RNA polymerase II and poly(A) polymerase enzymes.     

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.     

Intracellular levels of polyadenylated RNA and loss of vigour in germinating wheat embryos

Polyadenylated-RNA (Poly(A)⁺RNA) levels have been studied during the germination of wheat embryos of high viability but differing vigour. In high-vigour embryos imbibed at 20°C the level of poly(A)⁺RNA falls dramatically over the first hour of imbibition, then remains constant up to 3 h of imbibition before increasing rapidly to a level similar to that found in the quiescent state by 7 h of imbibition. Median-vigour embryos imbibed at 20°C show similar changes in poly(A)⁺RNA content but the initial decrease and subsequent increase in poly(A)⁺RNA levels are less marked. On imbibition at 10°C, the poly(A)⁺RNA content in high-vigour embryos decreases to a lesser extent during the first hour than at 20°C and the level increases more slowly over the next 6 h than during the same time period at 20°C. The level of poly(A)⁺RNA in medianvigour embryos remains constant over the first 4 h of germination and then falls to a level of about half that found in quiescent high-vigour embryos. Polyacrylamide gel electrophoresis of total-RNA samples shows that the polyadenylic acid (poly(A)) sequences occur in RNA species ranging in size from 35-7S. Polyacrylamide gel electrophoresis of isolated poly(A) sequences demonstrates the presence of two size classes of poly(A) in quiescent embryos, but at 20°C a more heterodisperse pattern appears by 2 h of imbibition. At 10°C, two size classes of poly(A) persist throughout the period studied in both high- and median-vigour embryos, although in median-vigour embryos the ratio of larger: smaller poly(A)-tail sizes decreases more rapidly than in high-vigour embryos.Abbreviations Poly(A) polyadenylic acid - poly(U) polyuridylic acid - poly(A)⁺RNA polyadenylated RNA     

Cell-free translation analysis of messenger RNA in echinoderm and amphibian early development

A wheat germ cell-free translation system has been used to analyze populations of abundant messenger RNA from sea urchin eggs and embryos and from amphibian oocytes and ovaries. We show directly that sea urchin eggs and embryos contain translatable mRNA of three general classes: poly(A)⁺ mRNA, poly(A)^? histone mRNA, and poly(A)^? nonhistone mRNA. Additionally, some histone synthesis appears to be promoted by poly(A)⁺ RNA. Sea urchin eggs seem to contain a higher proportion of prevalent poly(A)^? nonhistone mRNAS than do embryos. Some differences in the proteins encoded by poly(A)⁺ and poly(A)^? RNAs are detectable. Many coding sequences in the egg appear to be represented in both poly(A)⁺ and poly(A)^? RNAs, since the translation products of the two RNA classes exhibit many common bands when run on one-dimensional polyacrylamide gels. However, some of this overlap is probably due to fortuitous comigration of nonidentical proteins. Distinct stage-specific changes in the spectra of prevalent translatable mRNAs of all three classes occur, although many mRNAs are detectable throughout early development. Particularly striking is the presence of an egg poly(A)^? mRNA, encoding a 70,000–80,000 molecular weight protein, which is not detected in morula or later-stage embryos. In amphibian (Xenopus laevis and Triturus viridescens) ovary RNA, the translation assay detects the following three mRNA classes: poly(A)⁺ nonhistone mRNA, poly(A)^? histone mRNA, and poly(A)⁺ histone mRNA. Amphibian ovary RNA appearently lacks an abundant poly(A)^? nonhistone mRNA component of the magnitude detectable in sea urchin eggs. mRNA encoding histone-like proteins is found in the very earliest (small stage 1) oocytes of Xenopus as well as in later stage oocytes. During oogenesis there appear to be no striking qualitative changes in the spectra of prevalent translatable mRNAs which are detected by the cell-free translation assay.     

Messenger ribonucleoprotein particles in developing sea urchin embryos

Messenger RNA entering polysomes during early development of the sea urchin embryo consists of both oogenetic and newly transcribed sequences. Newly transcribed mRNA enters polysomes rapidly while oogenetic mRNA enters polysomes from a pool of stable, nontranslatable messenger ribonucleoprotein particles (mRNPs) derived from the unfertilized egg. Protein content may relate to differences in the regulation of newly transcribed and oogenetic mRNAs. Oogenetic poly(A)⁺ mRNA was found to be present in both polysomal and subpolysomal fractions of cleavage stage and early blastula stage embryos. This mRNA was found to be present in subpolysomal mRNPs with a density of 1.45 g/cm³ in Cs₂SO₄. Poly(A)⁺ mRNPs released from polysomes of embryos cultured in the presence of actinomycin D sedimented in a broad peak centered at 55 S and contained RNA of 21 S. The density of these particles was sensitive to the method of release; puromycin-released mRNPs had a density of 1.45 g/cm³, while EDTA caused a shift in density to 1.55 g/cm³, indicating a partial loss of protein. The results with newly synthesized mRNAs contrast sharply. Newly transcribed mRNA in subpolysomal mRNPs had a density of 1.55–1.66 g/cm³, a density approaching that of deproteinized RNA. Messenger RNA released from polysomes either by EDTA or puromycin was examined to determine the possible existence of polysomal mRNPs. When [³H]uridine-labeled mRNA was released from late cleavage stage embryo polysomes by either technique, and centrifuged on sucrose gradients, two broad peaks were found. One peak centered at 30 S contained 21 S mRNA while the other at 15 S contained 9 S histone mRNA. When these fractions were fixed with formaldehyde, they banded on Cs₂SO₄ gradients at a density of 1.60–1.66 g/cm³, very similar to that of pure RNA. We conclude that the newly transcribed mRNA may be present in stable mRNPs containing up to 10% protein in either subpolysomal or polysomal fractions. These mRNPs are clearly distinguishable from the protein-rich mRNPs containing oogenetic mRNAs.     

Differential accumulation of two size classes of poly(A) associated with messenger RNA during oogenesis in Xenopus laevis

The RNA of full-grown oocytes of Xenopus laevis contains two distinct size classes of poly(A), designated poly(A)_S and poly(A)_L, which contain 15–30 (mean = 20) and 40–80 (mean = 61) A residues, respectively. Both poly(A)_L and poly(A)_S are associated with RNA which is heterogeneous in size. The two classes of poly(A)⁺ RNA can be separated by affinity chromatography: Only poly(A)_L⁺ RNA binds to oligo(dT)-cellulose under appropriate conditions, but up to 50% of the poly(A)_S⁺ RNA can be isolated from the void fraction by binding to poly(U)-Sepharose. Both classes of poly(A)⁺ RNA are active as messenger RNA in an in vitro system and yield identical patterns of in vitro protein products. Previtellogenic oocytes contain almost exclusively poly(A)_L, which accumulates up to vitellogenesis but remains almost constant in amount (molecules/oocyte) during vitellogenesis and in the full-grown oocyte. Poly(A)_S accumulates (molecules/oocyte) from early vitellogenesis up to the full-grown oocyte. The total number of poly(A)⁺ RNA molecules per oocyte increases throughout oogenesis from 2 × 10¹⁰/previtellogenic oocyte [80–90% poly(A)_L] to 20 × 10¹⁰/full-grown oocyte (25–40% poly(A)_L). It is argued that poly(A)_S is protected from degradation in the oocyte, thus stabilizing the “maternal” poly(A)⁺ mRNA.     

Electrophoretic analysis of newly synthesized and stored maternal RNA during oogenesis ofCalliphora erythrocephala (Dipt.)

Summary Ovaries ofC. erythrocephala synthesize large amounts of poly(A)⁺ and poly(A)^– RNA during early and middle stages of oogenesis as shown by labelling with³H-uridine in vivo. After incubation for 1 h, a striking difference in the electrophoretic pattern of newly synthesized labelled poly(A)⁺ RNA and the poly(A)⁺ RNA present in sufficient amounts for optical density measurements (steady state poly(A)⁺ RNA) was observed. During early and mid-oogenesis, in the poly(A)^– RNA fraction, 4S predominantly mature rRNA, 5S RNA and tRNA were labelled. These fractions were no longer synthesized during late oogenesis, whereas poly(A)⁺ RNA was labelled continously During oogenesis stage specific differences in the size distribution of newly synthesized and steady state poly(A)⁺ RNA were not obvious. However, different sizes of labelled poly(A)⁺ RNA species were detected in 0–2h old preblastoderm embryos, after injection of³H-uridine into females either 3–4 days (stage 3–4 of oogenesis) or 24 h before oviposition (stage 5–6 of oogenesis). This difference in RNA synthesis was related to the presence of active nurse cell nuclei. The poly(A)⁺ RNA fraction represents about 2–3% of the total RNA in both ovaries and freshly laid eggs as judged by measurements of optical density and radioactivity bound to oligo(dT). The length of poly(A)-segments in ovarian poly(A)⁺ RNA varied from about 30 to 200 nucleotides.     

The size of poly(A)-containing RNAs in Drosophila melanogaster embryos

The size range of poly(A)-containing RNA from Drosophila melanogaster embryos has been estimated by hybridization with ³H-labeled poly(U) and subsequent fractionation on sucrose gradients. The median size of nuclear poly(A)-containing RNA is about 30 S (6000 nucleotides), and the median size of cytoplasmic poly(A)-containing RNA is about 17 S (1800 nucleotides). The relationship of these sizes to messenger RNA needed to code for protein and to the length of DNA contained in a chromomere is discussed.Research grant support was provided by NIH (6M35558; HD-00266) and NSF (GB-30600).     

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.