Ribosomal RNA Turnover in Contact Inhibited Cells

CONTACT inhibition of animal cell growth is accompanied by a decreased rate of incorporation of nucleosides into RNA^1–3. Contact inhibited cells, however, transport exogenously-supplied nucleosides more slowly than do rapidly growing cells^4,5, suggesting that the rate of incorporation of isotopically labelled precursors into total cellular RNA may be a poor measure of the absolute rate of RNA synthesis by these cells. Recently, Emerson⁶ determined the actual rates of synthesis of ribosomal RNA (rRNA) and of the rapidly labelled heterogeneous species (HnRNA) by labelling with ³H-adenosine and measuring both the specific activity of the ATP pool and the rate of incorporation of isotope into the various RNA species. He concluded that contact inhibited cells synthesize ribosomal precursor RNA two to four times more slowly than do rapidly growing cells, but that there is little if any reduction in the instantaneous rate of synthesis of HnRNA by the non-growing cells. We have independently reached the same conclusion from simultaneous measurements on the specific radioactivity of the UTP pool and the rate of ³H-uridine incorporation into RNAs (unpublished work of Edlin and myself). However, although synthesis of the 45S precursor to ribosomal RNA is reduced two to four times in contact inhibited cells, the rate of cell multiplication and the rate of rRNA accumulation are reduced ten times. This suggests either “wastage”⁷ of newly synthesized 45S rRNA precursor, or turnover of ribosomes in contact inhibited cells Two lines of evidence suggest that “wastage” of 45S RNA does not play a significant role in this system. (1) The rate of synthesis of 45S RNA in both growing and contact inhibited cells agrees well with that expected from the observed rates of synthesis of 28S and 18S RNAs (unpublished work of Edlin and myself). Emerson has made similar calculations⁶. (2) 45S RNA labelled with a 20 min pulse of ³H-uridine is converted in the presence of actinomycin D to 28S and 18S RNAs with the same efficiency (approximately 50%) in both growing and contact inhibited cells. These results indicate that, in order to maintain a balanced complement of ribosomal RNAs, contact inhibited cells must turn over their ribosomes. We present evidence here that rRNA is stable in rapidly growing chick cells, but begins to turn over with a half-life of approximately 35–45 h as cells approach confluence and become contact inhibited.     

Molecular Basis for Repressor Activity of Qβ Replicase

WITH the purification and characterization of viral replicases, a novel feature of nucleic acid polymerases—stringent template specificity—was recognized^1,2. Qβ replicase, the most extensively studied viral RNA polymerase^2–8, is now known to replicate Qβ RNA², the complementary Qβ minus strand⁹, RNA molecules described as “variants” of Qβ RNA^10,11 and a set of small RNAs of unknown origin which accumulate in Qβ-infected Escherichia coli, collectively designated as “6S RNA”¹². On the other hand, the RNA from phages related distantly, if at all, to Qβ^13,14, such as MS2 or R17 and of other viruses such as TMV² or AMV (Diggelmann and Weissmann, unpublished results) are completely inert as templates, as are ribosomal and tRNA from E. coli². Poly C and C-rich synthetic copolymers at high concentrations elicit synthesis which, however, remains restricted to the formation of a strand complementary to the template^15,16.     

Isolierung und Charakterisierung schnell markierter,hochmolekularer RNS aus frei suspendierten Calluszellen der Petersilie (Petroselinum sativum)

Summary By culturing of callus tissue originating from root explants of Petroselinum sativum in a synthetic liquid medium under aeration, freely suspended single cells and small clusters consisting of mostly five cells were obtained. The rapidly dividing cells did not exhibit any morphogenesis. Their nucleic acid metabolism was investigated by pulse experiments with ³²P-orthophosphate. Rapidly labelled RNA was prominently found associated with high molecular RNA. During the fractionation of the total nucleic acids on MAK columns it was eluted after the ribosomal RNA components. Its base ratio, however, differed from the latter in that the AMP content was higher than the GMP content. Sucrose gradient centrifugation and polyacrylamide gel electrophoresis resulted in the separation of the ribosomal RNA from the rapidly labelled RNA, thus proving the higher molecular weight of the latter. Based upon the migration in the gel a sedimentation coefficient of approximately 32S was calculated. The possible function of the heavy rapidly labelled RNA component as precursor of ribosomal RNA is discussed.     

Isolierung und Charakterisierung schnell markierter RNS von Euglena gracilis mit Hilfe analytischer und präparativer Elektrophorese in Polyacrylamid-Gelen

Zusammenfassung MAK-Säulenchromatographie der Gesamtnucleinsäuren aus autotrophen und gebleichten Zellen von Euglena gracilis, welche für 2 Std mit ³²P-Orthophosphat markiert wurden, liefert 6 Komponenten: niedermolekulare RNS (I–III), DNS (IV) und hochmolekulare RNS (V, VI). Das in der DNS-Region eluierte Material konnte mittels Gelfiltration in ³²P-DNS, in eine ³²P-RNS mit hoher spezifischer Aktivität sowie in ³²P-markierte Polyphosphate aufgetrennt werde. Außerdem fanden sich letztere in der ³²P-RNS-Fraktion, die relativ fest an die MAK-Säule gebunden bleibt. Eine weitaus bessere Auftrennung der einzelnen RNS-Komponenten gelang mit der Elektrophorese in Polyacrylamid-Gelen. So erschienen in 9.5% Gel 5 Komponenten, darunter die 3 niedermolekularen I–III, welche bei MAK-Chromatographie auftreten. Sie wurden als 4 S Transfer-RNS (I), 5 S ribosomale RNS (II) und 6 S RNS (III) identifiziert. Die hochmolekulare RNS wurde bei Auftrennung in 2,6% Gel in 6 Banden zerlegt. Die der ribosomalen RNS fanden sich als Hauptbanden in der 24 S und 20 S Region des Gels. Aufgrund ihrer Position konnten für die übrigen Komponenten Sedimentationskoeffizienten zwischen 18 S und 9 S berechnet werden. Das elektrophoretische Trennmuster der Gesamtnucleinsäuren aus gebleichten Zellen war sehr ähnlich, wenngleich quantitative Unterschiede zwischen den einzelnen Komponenten bestanden. Bei der Fraktionierung der Nucleinsäuren durch Gel-Elektrophorese im präparativen Maßstab fiel für jede markierte RNS-Komponente genügend Material an, um eine Rechromatographie an MAK und die Bestimmung der Basenzusammensetzung durchzuführen. Außer Transfer-RNS und 5 S RNS wurden 2 Komponenten in 9,5% Gel isoliert, deren Zusammensetzung ribosomaler RNS entsprach. Eine weitere niedermolekulare Komponente wurde als die schnell markierte RNS identifiziert, welche gemeinsam mit der DNS von der MAK-Säule eluiert wird. Die präparative Gel-Elektrophorese der ³²P-markierten hochmolekularen RNS in 2,6% Gel lieferte neben mehreren ribosomalen Species auch ³²P-RNS mit einer hohen spezifischen Aktivität.

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Isolation and characterization of rapidly labelled RNA from Euglena gracilis by means

Summary MAK column chromatography of total nucleic acids from autotrophic and bleached cells of Euglena gracilis cultured with ³²P_i for 2 h resulted in the separation of six labelled components: low molecular RNA (I–III), DNA (IV) and high molecular RNA (V, VI). Gel filtration of the material eluted in the DNA region revealed the presence of ³²P-RNA with a high specific activity and of ³²P-labelled polyphosphates in addition to ³²P-DNA. ³²P-polyphosphates were also found among the labelled RNA tenaciously bound to the MAK column. A far better resolution of the RNA components, however, was achieved by polyacrylamide-gel electrophoresis. On a 9.5% gel five main fractions were resolved among which appeared the components I–III isolated by MAK chromatography. They were identified as 4 S transfer RNA (I), 5 S ribosomal RNA (II) and 6 S RNA (III). The high molecular RNA gave rise to six bands when a 2.6% gel was used. From these the ribosomal RNA migrated as two bands in the 24 S and 20 S region of the gel. Based upon these values sedimentation coefficients from 18 S to 9 S were calculated for the others. The electrophoretic pattern of total nucleic acids from bleached cells was rather similar; only quantitative differences were observed. Fractionation of the nucleic acids by polyacrylamide-gel electrophoresis on a preparative scale provided enough material of each labelled RNA component to perform a rechromatography on MAK and to determine the base composition. Besides the 4 S transfer RNA and the 5 S RNA two RNA components with a ribosomal type base composition were isolated on a 9.5% gel. Another one was identified as the rapidly labelled RNA which is eluted with the DNA from the MAK column. Preparative gel electrophoresis of the labelled high molecular RNA (2.6% gel) revealed the presence of several ribosomal species in addition to ³²P-RNA components with a high specific activity.

     

Subunit interactions of rabbit muscle aldolase. Conformational change of aldolase in magnesium chloride and its relationship to the dissociation of subunits.

The effect of Escherichia coli ribosomal protein S1 on translation has been studied in S1-depleted systems programmed with poly(U), poly(A) and MS2 RNA³. The translation of the phage RNA depends strictly on the presence of S1. Optimum poly(U)-directed polyphenylalanine synthesis and poly(A)-programmed polylysine synthesis also require S1. Excess S1 relative to ribosomes and messenger RNA results in inhibition of translation of MS2 RNA and poly(U), but not of poly (A). In the case of phage RNA translation, this inhibition can be counteracted by increasing the amount of messenger RNA. Three other 30 S ribosomal proteins (S3, S14 and S21) are also shown to inhibit MS2 RNA translation. The effects of S1 on poly(U) translation were studied in detail and shown to be very complex. The concentration of Mg²⁺ in the assay mixtures and the ratio of S1 relative to ribosomes and poly(U) are crucial factors determining the response of this translational system towards the addition of S1. The results of this study are discussed in relation to recent developments concerning the function of this protein.     

Mutant Ribosomal Protein with Defective RNA Binding Site

THE 30S ribosomal subunits of Escherichia coli contain twenty-one different proteins^1–4, which together with 16S RNA can reassemble in vitro to form functional 30S particles⁵. Five proteins can individually bind to specific sites on the 16S RNA^6–8 and these are S4, S7, S8, S15 and S20 (in the nomenclature recently adopted by several laboratories to report results with the E. coli system⁹). We report here the first identification of a mutation that affects a ribosomal protein-nucleic acid interaction.     

Kern-Plasma-Transport neusynthetisierter rRNS in Zellen einer Suspensionskultur von Petroselinum sativum

Summary A rapidly labelled rRNA precursor can be detected in callus cells of Petroselinum sativum grown on a liquid synthetic medium. Its molecular weight has been calculated to be 2.3×10⁶. This value agrees with that of the rRNA precursor from other plant material. In order to follow the synthesis and processing of rRNA in time and to correlate single steps in this process with cell organelles it was necessary to obtain pure fractions of nuclei and ribosomes. The isolation method for nuclei is given in detail. The nucleic acids are separated on polyacrylamide gels of low acrylamide concentration. Pulse-chase experiments show that the rRNA precursor is split into two fragments within the nucleus: an 18S and a 25S component. The 18S RNA leaves the nucleus rapidly. It is already found quantitatively in the ribosomal fraction after 30–60 min chase. At that time the 25S RNA is still within the nucleus; it appears much later in the ribosomes. Since the increase in ribosomal label occurs simultaneously with the decrease in nuclear label, it is concluded that there is no degradation of 18S RNA within the nucleus. Apparently there are two distinct transport mechanisms with different kinetics for the two RNA components.     

Separation of α- and β-Globin Messenger RNAs

THE 10S RNA fraction of reticulocytes from various species contains the haemoglobin messenger RNA^1–4. When this 10S RNA fraction is added to a cell-free system derived from reticulocytes or Krebs II ascites cells, it directs the synthesis of α and β chains of haemoglobin^5–8. The α and β messenger RNA molecules contained in this fraction, however, have not yet been separated and identified. When reticulocyte. RNA of mouse is subjected to electrophoresis on 6% polyacrylamide gels, the 10S fraction contains two major bands and three minor bands⁹, suggesting that the major lOS RNA bands contain the messenger RNAs for the α- and β-globin chains.     

The absence of saturated pyrimidine bases in chromatin-associated RNA from avian reticulocytes and mouse ascites cells

Low molecular weight RNA (3–4S) has been isolated from the chromatin of avian reticulocytes and mouse ascites cells by the procedures used to isolated chromosomal RNA¹. Although dihydrouridine was readily detected in tRNA, neither this base, nor dihydroribothymidine, was found in detectable levels in chromatin-associated RNA or ribosomal RNA. The presence of saturated pyrimidines is not an invariant property of chromosomal RNA.     

STUDIES ON RAPIDLY LABELLED NUCLEAR RNA OF RAT BRAIN

—Methyl albumin kieselguhr chromatography (MAK) has been employed to separate rat brain nuclear RNA, labelled in vivo with [³H]uridine, into three major fractions. The first fraction (QI RNA) is ribosomal in nature for it has a high G + C/U ratio and is methylated by [methyl-³H] methionine. The other two fractions (Q2 RNA and TD RNA) are DNA-like for they exhibit a low G + C/U ratio and are labelled minimally by methionine. Pure ribosomal RNA chromatographs almost entirely in the Q1 RNA fraction. Labelling studies indicate that ribosomal RNA and DNA-like RNA behave differently. Initially, the label in the DNA-like RNA fractions increases rapidly and in a linear fashion for the first 30 min, but thereafter decreases rapidly and reaches a steady state level by 1 h and remains so up to at least the 2 h period. In contrast, the labelling of ribosomal RNA is much slower than that of DNA-like RNA during the first 30 min; however, unlike DNA-RNA, the labelling of ribosomal RNA still continues to increase linearly thereafter. Thus, during longer labelling periods, ribosomal RNA is labelled more rapidly than DNA-like RNA. It appears that the labelling of ribosomal RNA relative to DNA-like RNA is more rapid in liver than in brain.     

Synthèse des RNA au cours de la germination du radis

RNA synthesis in radish is studied during the first stages of germination. The radish seeds allowed to germinate in the dark, on distilled water, synthesize ribosomal RNA and accumulate a particular RNA, not incorporated in ribosomes. The results of ³²P incorporation in RNA of radish seedlings indicate a progressive formation of ribosomal RNA. Two species of rapidly labelled RNA are synthesized. With labelling time, their chromatographic behaviour on MAK columus evolves, while their electrophoretic characteristics remain stable. It is assumed that these two species are involved in ribosome formation. In vivo experiments with chloramphenicol support this conclusion. RNA which accumulates during germination, could be a particular type of ribosomal RNA which could be enable, under the definite culture conditions, to enter into ribosomal structures.     

Rate of Synthesis of Non-Ribosomal RNA in Castrate Uterus after Oestradiol-17β Stimulation

OESTROGENS have been reported to stimulate preferentially the synthesis of ribosomal RNA in the castrate uterus^1–4. Thus it has been suggested that 50% or more of the RNA that is synthesized in the oestrogen-stimulated uterus is ribosomal precursor RNA^1,2,4. The concept is supported by the reports of enhanced ribosome formation during early oestrogen action^5,6. It has also been shown, however, that during the first 6 h after oestrogen administration there is no increase in total uterine RNA in the rat uterus^4,7 and also the castrate mouse uterus⁸. These findings seem to be incompatable with the idea that much of the RNA that is synthesized during this first 6 h is ribosomal precursor RNA, most of which accumulates as new stable rRNA. Determination of the absolute rates of total RNA synthesis in vivo should provide some insight into the amounts of various species of RNA that are synthesized after oestrogen administration. Data presented here for the rate of total RNA synthesis strongly suggest that all except a small portion of the RNA that is being synthesized at 4 h after oestrogen stimulation is unstable in vivo and hence is not ribosomal precursor RNA.     

Studies on protein multimers. VII. Stabilization of E. coli beta-galactosidase by a 260-nm absorbing compound in crude preparations

5-Azacytidine, which has been shown to inhibit the maturation of ribosomal RNA from its precursors when added to the medium of cultured Novikoff hepatoma cells, alters the electrophoretic mobilities of the 45S and 32S ribosomal RNA precursors formed in Novikoff cells. Coelectrophoresis of total cell RNA samples shows that the 45S and 32S RNA precursors from 5-azacytidine-treated cells migrate slower than the corresponding precursors from control cells. 5-Azacytidine causes some reduction in the rate and degree of methylation of the 45S and 32S RNA precursors; however, the alteration in electrophoretic mobility and maturation of these two RNA species do not appear to be a consequence of undermethylation. Coelectrophoresis of undermethylated RNA produced by methionine starvation with normal RNA shows no differences in mobility of the ribosomal precursor RNA_s. 5-Fluorouridine and 8-azaguanine, also inhibitors of ribosomal RNA maturation, were not found to cause detectable differences in migration of the ribosomal precursor RNA_s.     

Nucleotide sequence of 5S RNA ofMycobacterium smegmatis

³²P labelled 5S RNA isolated fromMycobacterium smegmatis was digested withT ₁ and pancreatic ribonucleases separately and fingerprinted by two dimensional high voltage electrophoresis on thin-layer DEAE-cellulose plates. The radioactive spots were sequenced and their molar yields were determined. The chain length of the 5S RNA was found to be 120. It showed resemblances to both prokaryotic and eukaryotic 5S RNAs.     

RNA Polymerase III of <Emphasis Type="Italic">Blastocladiella emersonii</Emphasis> is Mitochondrial

MULTIPLE RNA polymerases have been shown to exist in a wide variety of eukaryotic organisms^1–5. Two nuclear polymerases have been found in all the cells studied, each with a specific location and a specific function: the DEAE fraction I enzyme is located in the nucleolus and may be involved in the synthesis of ribosomal RNA^1,2,5,6; the DEAE fraction II enzyme is located in the non-nucleolar nucleoplasm and functions in the synthesis of DNA-like RNA^2–5,7. The DEAE fraction III enzyme was reported to exist in sea urchin¹, the aquatic fungus B. emersonii⁵ and to be present sometimes in rat liver preparations^1,8. Although there have been some reports that polymerase III is nuclear, Horgen and Griffin⁵ showed that the enzyme was sensitive to the prokaryotic RNA polymerase inhibitor rifampicin. They suggested that the fraction III enzyme may be mitochondrial, formed as the result of organelle contamination in their crude nuclear preparations. The results of this study show that the DEAE fraction III enzyme in B. emersonii is a mitochondrial enzyme, most likely functioning in the synthesis of mitochondrial RNA. The rifampicin sensitivity of the enzyme is further evidence of a prokaryotic origin of mitochondria^9,10.     

Detection of specific sequences among DNA fragments separated by gel electrophoresis.

Ribosomal RNA and precursor ribosomal RNA from at least one representative of each vertebrate class have been analyzed by electron microscopic secondary structure mapping. Reproducible patterns of hairpin loops were found in both 28 S ribosomal and precursor ribosomal RNA, whereas almost all the 18 S ribosomal RNA molecules lack secondary structure under the spreading conditions used. The precursor ribosomal RNA of all species analyzed have a common design. The 28 S ribosomal RNA is located at or near the presumed 5′-end and is separated from the 18 S ribosomal RNA region by the internal spacer region. In addition there is an external spacer region at the 3′-end of all precursor ribosomal RNA molecules. Changes in the length of these spacer regions are mainly responsible for the increase in size of the precursor ribosomal RNA during vertebrate evolution. In cold blooded vertebrates the precursor contains two short spacer regions; in birds the precursor bears a long internal and a short external spacer region, and in mammals it has two long spacer regions. The molecular weights, as determined from the electron micrographs, are 2·6 to 2·8 × 10⁶ for the precursor ribosomal RNA of cold blooded vertebrates, 3·7 to 3·9 × 10⁶ for the precursor of birds, and 4·2 to 4·7 × 10⁶ for the mammalian precursor. Ribosomal RNA and precursor ribosomal RNA of mammals have a higher proportion of secondary structure loops when compared to lower vertebrates. This observation was confirmed by digesting ribosomal RNAs and precursor ribosomal RNAs with single-strandspecific S₁ nuclease in aqueous solution. Analysis of the double-stranded, S₁-resistant fragments indicates that there is a direct relationship between the hairpin loops seen in the electron microscope and secondary structure in aqueous solution.     

Characteristics and intracellular distribution of messengerlike RNA in encysted embryos of Artemia salina

Homogenates of dormant cysts of Artemia salina were fractionated by differential centrifugation. RNA was prepared from the various fractions and tested for stimulatory activity in a [¹⁴C]leucine incorporating Escherichia coli system. The highest specific activity was found in the RNA extracted from a cytoplasmic fraction sedimenting at 15,000 g. Some activity was associated with the soluble and crude ribosomal fractions, while the RNA extracted from the crude nuclear fraction was less active.The 15,000 g sediment was purified by centrifugation in a sucrose density gradient. The active material formed a characteristic, colored band at a buoyant density of about 1.17 g/ml. The banding fraction was mainly composed of endoplasmic vesicles and mitochondria. The specific activity of the extracted RNA was further increased when the 15,000 g sediment was treated with buffered 20–100 mM EDTA (with or without 0.1% Triton X-100) before banding.Sedimentation analysis of the active RNA from the purified 15,000 g fractions revealed three distinct absorption peaks at 28 S, 18 S, and 16 S, apparently representing cytoplasmic and mitochondrial rRNA. The 28 S and 18 S peaks were reduced by EDTA treatment, but only to a certain limit. By gel electrophoresis a number of additional components were resolved, including 4 S and 5 S RNA. The template activity showed a heterodisperse distribution with a maximum at 17–20 S, not correlated with the 16 S peak. Isolated 18 S and 28 S rRNA had very low activity.The experiments suggest that in Artemia cysts an appreciable amount of messengerlike RNA is associated with mitochondria and/or endoplasmic vesicles carrying ribosomal monomers.     

Vorstufen der ribosomalen RNA in frei suspendierten Calluszellen der Petersilie (Petroselinum sativum)

Summary Six high molecular weight, rapidly labelled RNA species were detected in freely suspended callus cells of Petroselinum sativum by means of isotope labelling and electrophoretic separation in agarose-polyacrylamide gels. On the basis of their migration in the latter the RNA species were calculated to have the following molecular weights: 2.9×10⁶, 2,4×10⁶, 1.9×10⁶, 1.4×10⁶, 1.0×10⁶ and 0.75×10⁶ daltons. Thus they can clearly be distinguished from the two ribosomal RNA species (1.3×10⁶ and 0.7×10⁶ daltons). During incubation of the cells with [³H]methyl-methionine as a methyl donator all six components incorporated radioactivity rapidly. With [³H]nucleosides or [³H]orotic acid as precursors the 2.9×10⁶ and the 2.4×10⁶ daltons RNA were labelled within 10 min, while the other high molecular weight species appeared after about 20 min of labelling.Prolongation to 45–120 min resulted in accumulation of radioactivity preferentially in the 1.4×10⁶ and 0.75×10⁶ daltons RNA and in the ribosomal RNA species. The results of cell fractionation experiments provide evidence that these rapidly labelled high molecular weight RNA species are synthesized in the cell nucleus. The kinetics of their synthesis together with the other data obtained strongly support the suggestion that these RNA species function as precursors in the processing of ribosomal RNA. The possible mechanism of this process is discussed.

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Verwendete Abkürzungen EDTA Äthylendiamintetraessigsäure - DNase Desoxyribonuclease - Imp./min epm - MAK methyliertes Albumin an Kieselgur - POPOP 1,4- bis (4-Methyl-5-Phenyloxazol)-Benzol - PPO 2,5-Diphenyloxazol - RNase Ribonuclease - S Sedimentationskoeffizient in Svedberg-Einheiten - SDS Natriumdodecylsulfat - TPE Tris-Phosphat-EDTA-Puffer - Tris Tris-(hydroxymethyl)-aminomethan - Upm rpm     

In vitro synthesis of RNA by Xenopus spermatogenic cells I. Evidence for polyadenylated and non-polyadenylated RNA synthesis in different cell populations.

Premeiotic and postmeiotic (haploid) gene expression during spermatogenesis in the anuran, Xenopus laevis, was studied by analyzing the accumulation of radioactively labelled cytoplasmic polyadenylated [poly (A +)] and non-polyadenylated [poly (A -)] RNAs. Dissociated spermatogenic cells were labelled and maintained in an in vitro system capable of supporting cell differentiation. Labelled cells were separated by density gradient centrifugation into subpopulations enriched for individual spermatogenic stages. RNA was extracted and purified from each cell fraction, and separated into poly (A +) and poly (A -) species. Comparison of poly (A +) to non-poly (A) radioactivity in cells labelled with tritiated uridine or adenosine demonstrated that (1) all cell fractions produced significant quantities of polyadenylated RNA relative to total RNA synthesis; and (2) that a cell fraction enriched for pachytene spermatocyte RNA contained up to 15% of total cytoplasmic and 35% of total polysomal RNA labelled as poly (A +) containing species. RNA was also characterized by sucrose density gradient centrifugation and polyacrylamide gel electrophoresis. All cell types showed typical poly (A -) peaks of 4S, 18S and 28S, corresponding to tRNA (4S) and rRNAs (18, 28S) respectively. Spermatids and spermatozoa had additional absorbance peaks at 13 and 21S which cosedimented with Xenopus oocyte mitochondrial rRNA. Patterns of incorporation of uridine and adenosine into poly (A +) RNA in all germ cell fractions tested were complex. In all cases, major areas of radioactivity were found in a broad band sedimenting between 6-17S. Spermatid fractions showed a prominent peak of incorporation at 6-8S, while pachytene cells also showed heavier poly (A +) peaks in the 17-25S region. A non-polyadenylated RNA species sedimenting at 6-8S with a relatively rapid rate of turnover was also observed in spermatids. From these results it is concluded that synthesis of transfer, ribosomal, and putative messenger RNA species continues in spermatogenic cells throughout all but the very last stages of spermatogenesis in Xenopus.