Balbiani ring nucleotide sequences in cytoplasmic 75 S RNA of Chironomus tentans salivary gland cells

The giant puffs, the Balbiani rings (BR) 1 and 2 of Chironomus tentans polytene chromosomes synthesize large RNA molecules sedimenting at about 75S. An RNA fraction of approximately the same size is present in nuclear sap and cytoplasm. In situ hybridization of cytoplasmic 75S RNA and other electrophoretically defined cytoplasmic RNA fractions showed BR RNA to be confined to the 75S RNA, and absent in other high molecular-weight cytoplasmic RNA fractions, which indiates that BR RNA is transferred from the nucleus into the cytoplasm without an appreciable size reduction.     

NUCLEOLAR-DERIVED RIBONUCLEIC ACID IN CHROMOSOMES, NUCLEAR SAP, AND CYTOPLASM OF CHIRONOMUS TENTANS SALIVARY GLAND CELLS

The distribution of monodisperse high molecular weight RNA (38, 30, 28, 23, and 18S RNA) was studied in the salivary gland cells of Chironomus tentans. RNA labeled in vitro and in vivo with tritiated cytidine and uridine was isolated from microdissected nucleoli, chromosomes, nuclear sap, and cytoplasm and analyzed by electrophoresis on agarose-acrylamide composite gels. As shown earlier, the nucleoli contain labeled 38, 30, and 23S RNA. In the chromosomes, labeled 18S RNA was found in addition to the 30 and 23S RNA previously reported. The nuclear sap contains labeled 30 and 18S RNA, and the cytoplasm labeled 28 and 18S RNA. On the basis of the present and earlier analyses, it was concluded that the chromosomal monodisperse high molecular weight RNA fractions (a) show a genuine chromosomal localization and are not due to unspecific contamination, (b) are not artefacts caused by in vitro conditions, but are present also in vivo, and (c) are very likely related to nucleolar and cytoplasmic (pre)ribosomal RNA. The 30 and 23S RNA components are likely to be precursors to 28 and 18S ribosomal RNA. The order of appearance of the monodisperse high molecular weight RNA fractions in the nucleus is in turn and order: (a) nucleolus, (b) chromosomes, and (c) nuclear sap. Since both 23 and 18S RNA are present in the chromosomes, the conversion to 18S RNA may take place there. On the other hand, 30S RNA is only found in the nucleus while 28S RNA can only be detected in the cytoplasm, suggesting that this conversion takes place in connection with the exit of the molecule from the nucleus.     

Transport of different RNA species from the nucleus to the cytoplasm in Xenopus laevis neurula cells

Isolated cells from Xenopus laevis neurulae were labeled, and the RNAs extracted from their nuclear and soluble cytoplasmic fractions were analyzed on polyacrylamide gels. In the soluble cytoplasm, 4S RNA emerged very rapidly, and this was immediately followed by the emergence of poly(A)-containing RNA and 18S ribosomal RNA. In contrast, the emergence of 28S ribosomal RNA was delayed by about 2 hr. The size distribution of cytoplasmic poly(A)-containing RNA was much smaller as compared to that of nuclear poly(A)-containing RNA. These results indicate that the newly synthesized RNAs in Xenopus neurula cells are transported from the nucleus to the cytoplasm in a characteristic sequence.     

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.     

Isolation and characterization of oat globulin messenger RNA

When polyadenylated RNA, isolated from membrane-bound polysomes extracted from developing oat (Avena sativa L.) seeds, was translated in vitro in the rabbit reticulocyte system, two polypeptides of about 58 and 60 kilodaltons were immunoprecipitated by anti-oat globulin antibody. No electrophoretic bands corresponding to the 40 and 20 kilodalton polypeptides of oat globulin were present. However, when in vivo labeled extracts were immunoprecipitated with anti-oat globulin antibody, three groups of polypeptides (60, 40, and 20 kilodaltons) were present. It therefore seems probable that the two large polypeptides (58 and 60 kilodaltons) were precursors of the 40 and 20 kilodalton polypeptides. When the polyadenylated RNA coding for these polypeptides was size fractionated on a sucrose density gradient, it sedimented near the 18S region of the gradient. Translation of the RNA from the gradient fractions and immunoprecipitation of translation products indicated that the template for the 58 to 60 kilodalton `putative' precursors of oat globulin was probably the RNA which was approximately 18S in size.     

RNA synthesis in the ovary and oocytes of the starfish

Qualitative studies on the in vitro uptake and incorporation of tritiated uridine into RNA of the somatic and germinal elements of the starfish ovary were carried out prior to and during hormone-induced oocyte maturation and spawning.Autoradiography of nonhormone-treated ovaries indicated that the outer ovarian wall contained the highest concentration of label, with lesser amounts in the follicle cells and least in the oocytes. Oocytes and follicle cells localized at the periphery of the ovary were labeled first, and both cells became progressively labeled throughout the ovary with time; the label first appeared localized in the nucleolus of the oocyte.Sucrose gradient analysis of the separated cellular components of prelabeled hormone-treated ovaries indicated that RNA synthesis occurred in all segments of the ovary and that the spawned oocyte fraction was the least active. Synthesis of ribosomal RNA was detectable after a lag period of approximately 4 hr. Oocytes incubated in ³H-uridine during and subsequent to 1-methyladenine-induced spawning and maturation synthesized 15–19 S and low molecular weight RNA but not ribosomal RNA. Synthesis of the 15–19 S RNA was inhibited with ethidium bromide and to a limited extent by actinomycin D. Isolated mitochondrial fractions contained most of the labeled 15–19 S RNA. These data suggest the mitochondrial origin of most, if not all, of this intermediate-weight RNA. On the basis of these studies, it appears that starfish oocytes and follicle cells are metabolically active at the transitional period from growth to maturational stages in oocytes. Synthesis of RNA furthermore apparently continues in the cytoplasm subsequent to germinal vesicle breakdown and spawning.     

Cytoplasmic methylation of mature 5.8 S ribosomal RNA

Levels of 2-O-methylation were determined in ribosomal 5·8 S RNAs from whole cells and both the nuclear and cytoplasmic fractions of rat liver, rat kidney cells in culture (NRK) and HeLa cells. All 5·8 S RNA molecules contained the alkali stable Gm-Cp dinucleotide at position 77 but only whole cell rat liver RNA contained large amounts (0·7 mol) of Um at position 14. All nuclear 5·8 S RNA fractions were largely undermethylated at this site. In contrast, cytoplasmic 5.8 S RNA from rat liver and, to a lesser degree, NRK cells contained significantly more Um; up to 80% of the molecules from rat liver contained the methylated residue. These results indicate that mature 5·8 S RNA can be methylated in the cytoplasm. When labeling kinetics were examined in NRK cells, the methylation at residue 14 was found to increase as a function of the time spent in the cytoplasm, confirming that this modification is, unlike other ribosomal RNA methylations, in part or largely cytoplasmic.     

A comparative study of RNA fractions mediating delayed sensitivity to a chemically defined antigen in vitro.

Hot-cold phenol extracts of RNA prepared from guinea pigs sensitized to mono (p-azobenzene-arsonate)-N-chloroacetyl-l-tyrosine (ARSNAT) contains limited but distinct fractions able to transfer ARSNAT or KLH sensitivity to guinea pig peritoneal exudate cells in vitro. Each of these fractions have been compared by oligo(dT) affinity chromatography and sucrose density gradient analysis. One RNA fraction initially obtained from a sucrose density gradient (designated as B fraction) possessed two separate peaks and contained polyadenylic acid sequences as evidenced by its ability to bind to an oligo (dT) column. Another fraction (Fraction II) initially isolated by oligo (dT) affinity chromatography possessed two peaks after sucrose density gradient analysis, contained poly-A sequences, and had an S-value range approximating the B fraction. RNA fractions prepared from the liver or skeletal muscle of sensitized guinea pigs fails to transfer ARSNAT sensitivity and all fractions are completely inactivated by bovine pancreatic RNase. The results suggest that portions of density gradient prepared B fraction and Fraction II binding to oligo (dT) cellulose may represent the same and/or similar moieties of immunobiologically active RNA.     

Hybridizable ribonucleic acid of rat brain

1. Cerebral RNA of adult and newborn rats was labelled in vivo by intracervical injection of [5-³H]uridine or [³²P]phosphate. Hepatic RNA of similar animals was labelled by intraperitoneal administration of [6-¹⁴C]orotic acid. Nuclear and cytoplasmic fractions were isolated and purified by procedures involving extraction with phenol and repeated precipitation with ethanol. 2. The fraction of pulse-labelled RNA from cerebral nuclei that hybridized to homologous DNA exhibited a wide range of turnover values and was heterogeneous in sucrose density gradients. 3. Base composition of the hybridizable RNA was similar to that of the total pulse-labelled material; both were DNA-like. 4. Pulse-labelled cerebral nuclear RNA hybridized to a greater extent than cytoplasmic RNA for at least a week after administration of labelled precursor. This finding suggested that cerebral nuclei contained a hybridizable component that was not transferred to cytoplasm. 5. The rates of decay of the hybridizable fractions of cerebral nuclei and cytoplasm were faster in the newborn animal than in the adult. Presumably a larger proportion of labile messenger RNA molecules was present in the immature brain. 6. Cerebral nuclear and cytoplasmic RNA fractions from newborn or adult rats, labelled either in vivo for periods varying from 4min. to 7 days or in vitro by exposure to [³H]-dimethyl sulphate, uniformly hybridized more effectively than the corresponding hepatic preparation. These data suggested that a larger proportion of RNA synthesis was oriented towards messenger RNA formation in brain than in liver.     

On the interpretation of small-angle x-ray solution scattering from spherical viruses.

The intermediates in the ribosome assembly in exponentially growing Escherichia coli have been identified by centrifuging a crude lysate, pulse-labeled with a radioactive RNA base, through a sucrose gradient and analyzing for precursor rRNA in the gradient fractions by gel electrophoresis. The major intermediate in the assembly of the 50 S subunit cosediments with the mature subunit, whereas two minor precursor species sediment between the 30 S and 50 S peaks. The assembly of the 30 S subunit proceeds via a minor intermediate sedimenting slightly behind the mature subunit and a major precursor particle that cosediments with the mature 30 S subunit.The fraction of the rRNA contained in these precursor particles was determined by direct determination of the amount of rRNA in the precursor particles, and from the labeling kinetics of their rRNA. The direct estimation indicated that about 2% of the total 23 S type RNA, and 3 to 5% of the total 16 S type RNA is harboured in precursor particles. In the kinetic experiments the specific activity of the nucleoside triphosphates and of the different ribosomal particles was followed after addition of a radioactive RNA precursor to the growth medium. The results were compared with a digital simulation of the flow of isotopes through the assembly pathways. This method indicated that approximately 2% of the total 23 S type RNA, as well as 2% of the total 16 S type RNA, is contained in the precursor particles.     

Rat liver chromatin. Fractionation into eu- and heterochromatin with localization of ribosomal genes.

Purified, normal rat liver chromatin was sheared under controlled conditions in a Virtis “60” homogenizer and then separated into template-active (euchromatin) and template-inactive (heterochromatin) fractions by glycerol gradient centrifugation. The euchromatin portions of the glycerol gradients possessed greater than 90% of the in vitro template activity when estimated with Escherichia coli RNA polymerase and contained more than 90% of the nascent RNA formed in vivo. Inhibitor studies performed in vivo indicated that the leading edge of the heterochromatin portion of the glycerol gradients contains the genes coding for ribosomal RNA. Recentrifugation of putative eu- and heterochromatin fractions demonstrated that the isolation procedure produces fractions which are distinct and are characterized by little or no cross contamination. The data suggest that controlled shearing in conjunction with gradient centrifugation provides a means of fractionating with great fidelity the transcribable portions of the rat liver genome.     

Intracellular transport of nuclear ribosomal RNA in Acetabularia mediterranea]

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

Synthesis and intercellular transport of ribosomal RNA in the ovary of the milkweed bug Oncopeltus fasciatus

Analysis of cellular and subcellular fractions obtained by dissection and subsequent purification of the main components of the Oncopeltus fasciatus ovary reveals that the uptake and incorporation of tritiated uridine into high molecular weight RNA differs markedly between the trophic syncytium and the oöcytes. The pattern of labelling indicates that the trophic syncytium is actively engaged in the synthesis of ribosomal RNA, but that the oöcytes do not synthesize detectable amounts of ribosomes. The ribosomal RNA which eventually appears in the cytoplasm of the oöcytes is postulated to come from the trophic syncytium via the nutritive cords. There is no other fraction of non-4 S RNA detectable in the oöcytes by these methods.     

Intracellular distribution of low molecular weight RNA in Chironomus tentans

Low molecular weight RNA species are described in isolated nuclear components and cytoplasm of salivary gland cells of Chironomus tentans. In addition to 4S and 5S RNA and RNA in the 4–5S range previously described, at least three other components in the range below 16S are present. RNA, the molecular weight of which was estimated to 2.3 x 10⁵ and designed 10S RNA, can be observed only in nucleoli; other RNA, the molecular weight of which was estimated to 1.3 x 10⁵ and designed 8S RNA, was detected in the chromosomes, the nuclear sap, and the cytoplasm but not in the nucleoli; and a third type of RNA, the molecular weight of which was estimated to 8.5 x 10⁴ and designed 7S RNA, was present in nucleoli, chromosomes, nuclear sap, and cytoplasm. The substituted benzimidazole, 5,6-dichloro-1 (β-D-ribofuranosyl)benzimidazole (DRB), which gives a differential inhibition of the labeling of heterodisperse, mainly high molecular weight RNA in the chromosomes, does not inhibit the labeling of 8S RNA. The relative amounts of label in 8S RNA and 4–5S RNA (including 4S RNA and 5S RNA) in different isolated chromosomes, are distributed in proportion to the chromosomal DNA contents. The 8S RNA as well as the 7S RNA show a relative accumulation in chromosomes and nuclear sap with prolonged incubation time and are in this respect similar to intranuclear low molecular weight RNA species described by previous workers. Our data suggest, however, that these two types of RNA may differ in an important aspect from the previously described types since they are also present in the cytoplasm.     

RNA from callus cultures and leaves of Nicotiana tabacum

MAK column chromatography has been used to analyse RNA from normal and crown gall callus cultures and leaves of Nicotiana tabacum. To determine the elution behaviour of well-defined DNA-like RNAs with different GC content, complementary RNAs (c-RNA) synthesized on Agrobacterium tumefaciens DNA and crown gall DNA were used. The elution profile of the RNA from all three tissues followed a similar pattern. By salt gradient elution the RNA in the tRNA region showed a remarkably high CMP content which was significantly higher for the normal tissues than for crown gall tissue. RNA from the callus cultures contained more DNA-like RNA (D-RNA) with a higher turnover rate than RNA from leaves. Because of its relatively low poly A content, measured as RNase A + T₁ resistance, as well as its high turnover rate, the salt-eluted D-RNA is thought to be heterogeneous nuclear RNA (Hn-RNA) and not mRNA. RNA molecules that might represent the mRNA population, having intramolecular poly A tracts, were subsequently eluted by a salt gradient, a low salt buffer and with the chaotropic agent guanidine thiocyanate, which removed tenaciously bound (TB-RNA) in two fractions, α and β. Crown gall RNA showed both a different labelling behaviour and a higher poly A content in the α and β fractions compared to the normal tissues. c-RNAs may be eluted at different salt concentrations because of their different GC content. They give rise to a considerable fraction of TB-RNA which in the presence of tobacco leaf RNA was split into fractions similar to α and β. No fraction was found amongst these RNAs which did have intramolecular poly A tracts.     

RIBOSOME SYNTHESIS IN TETRAHYMENA PYRIFORMIS

The cellular site of synthesis of ribosomal RNA in Tetrahymena pyriformis was studied by analyzing the purified nuclear and cytoplasmic RNA from cells pulse labeled with uridine-³H. The results of studies using zonal centrifugation in sucrose density gradients show that the ribosomal RNA is synthesized in the nucleus as a large precursor molecule sedimenting at 35S. The 35S molecule undergoes rapid transformation through two main nuclear intermediates, sedimenting at about 30S and 26S. The smaller ribosomal RNA (17S) appears first in the cytoplasm and it seems to be absent from the nucleus. The apparent delay in the appearance of the larger ribosomal RNA (26S) in the cytoplasm is due to the presence of a larger pool of its precursors in the nucleus as indicated by pulse-chase experiments. The newly synthesized ribosomal RNA's appear in the cytoplasm as discrete 60S and 45S ribonucleoprotein particles, before their incorporation into the polysomes.     

STUDIES ON ISOLATED NUCLEI : II. Isolation and Chemical Characterization of Nucleolar and Nucleoplasmic Subfractions

This paper describes the subfractionation of nuclei isolated from guinea pig liver by the procedure presented in the first article of the series (8). Centrifugation in a density gradient system of nuclear fractions disrupted by sonication permits the isolation of the following subfractions: (a) a nucleolar subfraction which consists mainly of nucleoli surrounded by a variable amount of nucleolus-associated chromatin and contaminated by chromatin blocks derived primarily from von Kupffer cell nuclei; (b) and (c), two nucleoplasmic subfractions (I and II) which consist mainly of chromatin threads in a coarser (I) or finer (II) degree of fragmentation. The protein, RNA, and DNA content of these subfractions was determined, and their RNA's characterized in terms of NaCl-solubility, nucleotide composition, and in vivo nucleotide turnover, using inorganic ₃₂P as a marker. The results indicate that there are at least three types of RNA in the nucleus (one in the nucleolus and two in the nucleoplasm or chromatin), which differ from one another in NaCl-solubility, nucleotide composition, turnover, and possibly sequence. Possible relations among these RNA's and those of the cytoplasm are discussed.     

Characterization of two plasmacytoma fractions and their RNA capable of changing lymphocyte surface immunoglobulins (cell conversion).

The cellular origin and the physicochemical characteristics of MOPC-300 RNA that has been found to induce changes in lymphocyte surface immunoglobulins (cell conversion) in normal mice were investigated. The RNA active in cell conversion was found associated with a particulate cytoplasmic fraction of the tumor that could be collected by centrifugation at 100,000g for 30 min. A particulate fraction with cell-converting activity was isolated also from the plasma of tumor-bearing animals. The plasma fraction had a buoyant density intermediate between 1.08 and 1.18 g/ml. Both active fractions migrate in the same electrophoretical region. RNA extracted from both the subcellular and plasma fraction, when analyzed on a sucrose gradient, yielded two populations of RNA molecules with cell-converting activity (14–18S and 40–50 S, respectively). The 40–50 S RNA was shown to be thermolabile. The RNA with cell-converting activity contained poly A stretches. The possible function of this RNA during the growth of the plasmacytoma is discussed.     

The Kinetics of the Synthesis of Ribosomal RNA in E. coli

The kinetics of the synthesis of ribosomal RNA in E. coli has been studied using C¹⁴-uracil as tracer. Two fractions of RNA having sedimentation constants between 4 and 8S have kinetic behavior consistent with roles of precursors. The first consists of a very small proportion of the RNA found in the 100,000 g supernatant after ribosomes have been removed. It has been separated from the soluble RNA present in much larger quantities by chromatography on DEAE-cellulose columns. The size and magnitude of flow through this fraction are consistent with it being precursor to a large part of the ribosomal RNA.

A fraction of ribosomal RNA of similar size is also found in the ribosomes. This fraction is 5 to 10 per cent of the total ribosomal RNA and a much higher proportion of the RNA of the 20S and 30S ribosomes present in the cell extract. The rate of incorporation of label into this fraction and into the main fractions of ribosomal RNA of 18S and 28S suggests that the small molecules are the precursors of the large molecules. Measurements of the rate of labeling of the 20, 30, and 50S ribosomes made at corresponding times indicate that ribosome synthesis occurs by concurrent conversion of small to large molecules of RNA and small to large ribosomes.

     

Balbiani ring RNA in the cytoplasm of Chironomus thummi salivary gland cells

In this paper experimental results on the size, transport and stability of cytoplasmic Balbiani ring RNA and on its appearance in polysomes are presented. Cytoplasmic RNA of salivary gland cells from Chironomus thummi contains two large RNA fractions of about 20×10⁶ dalton and 10×10⁶ dalton in size. These RNA fractions correspond both to Balbiani ring BR 1 RNA and BR 2 RNA and are apparently transported from nucleus into cytoplasm without a significant size reduction. Chase experiments illustrate a great stability of giant cytoplasmic Balbiani ring RNA molecules and exclude the possibility of a precursor-product relationship between these and smaller BR RNA molecules also found in cytoplasm. A part of giant cytoplasmic Balbiani ring RNA molecules is bound to poly(U)-sepharose columns and should, therefore, contain poly(A)-sequences. — Polysomes of salivary gland cells extracted by a gentle lysis procedure and centrifuged through sucrose gradients are characterized by a rather broad sedimentation profile. Polysome sizes up to about 800 S have been detected, but in no case a distinct polysome fraction corresponding in size to Balbiani ring RNA has been observed. Hybridization of polysomal RNA with salivary gland chromosomes in situ resulted in labelling of both Balbiani rings BR 1 and BR 2.