Characterization of ribonucleic acids from the venom glands of Crotalus durissus terrificus (Ophidia, Reptilia) after manual extraction of the venom. Studies on template activity and base composition

RNA synthesis in the venom glands of Crotalus durissus terrificus was stimulated by the manual extraction of the venom (milking). RNA was extracted from venom glands activated by milking and fractionated by centrifugation through sucrose density gradients. Template activity for protein synthesis and base composition of the RNA fractions were studied. RNA fractions that sediment between 18S and 4S had the highest template activity. The base composition analysis indicated that the 28S and 18S rRNA have a C+G content of 65.4 and 58% respectively. The ;melting' temperature (T(m)) of DNA in 0.15m-NaCl-0.015m-trisodium citrate, pH7.0, was 85 degrees C, corresponding to a C+G content of 38%. The base ratio of the RNA fractions that showed a high template activity was intermediate between that of rRNA and homologous DNA. The possible role of these fractions in the synthesis of the two main toxins (crotoxin and crotamine) of the South American rattlesnake's venom is discussed.     

Thermolability of 28S ribosomal ribonucleic acid from the liver of Crotalus durissus terrificus (Ophidia, Reptilia)

Instability of 28S rRNA of Crotalus durissus terrificus liver was observed during hotphenol extraction: purified 28S rRNA is converted into an 18S RNA component by heat treatment. It was also found that ;6S' and ;8S' low-molecular-weight RNA species were released during the thermal conversion. This conversion and the release of the low-molecular-weight species were also induced by 8m-urea and 80% (v/v) dimethyl sulphoxide at 0 degrees C. Evidence is presented that this phenomenon is an irreversible process and results from the rupture of hydrogen bonds. The 18S RNA product was shown to be homogeneous by polyacrylamide-gel electrophoresis and by sucrose-density-gradient centrifugation. The base composition of the 18S RNA products obtained by heat, urea or dimethyl sulphoxide treatments was similar. The C+G content of the 18S RNA product was different from that of the native 18S rRNA, but similar to that of 28S rRNA.     

FRACTIONATION OF THE RNA COMPONENTS OF RAT BRAIN POLYSOMES

Abstract— The incorporation in vivo of [³H]uridine into the RNA isolated from the free polyribosomes of rat cerebral cortex was studied. Sedimentation in sucrose gradients showed that initially (at times less than 60 min after injection of precursor) the label was associated with a heterodisperse species, while at longer times there was an increased coincidence of label with stable rRNA. Further fractionation was accomplished by means of differential extraction with phenol and analysis on polyacrylamide-agarose gels. Most of the rapidly labelled RNA was concentrated in a fraction obtained at pH 8-3 and 40°C. The base composition of this fraction differed greatly from that of rRNA, preribosomal RNA and DNA. Analysis by electrophoresis on polyacrylamide-agarose gels showed it to be composed of several distinct species in addition to residual 18 and 28S rRNA. Most of the latter was concentrated in a fraction extracted at pH 60 at 0°C.     

Studies on ribonucleic acid synthesis in the venom glands of Vipera palaestinae (Ophidia, Reptilia)

RNA metabolism in the venom glands of Vipera palaestinae was studied at different stages after manual extraction of the venom (milking). The rate of (32)P incorporation into gland RNA was found to be maximal at 1-4 days after milking in correlation with the height of the secretory epithelium. Venom production attained a maximum only after 8-16 days, in parallel with the accumulation of stable species of cellular RNA.     

The synthesis of ribonucleic acid in immature rat uterus responding to oestradiol-17β

Stimulation of incorporation of labelled precursors into the RNA of immature rat uterus is an early result of oestradiol-17beta action. However, the extent of the increased incorporation varies with the mode of administration of the labelled precursors and with the weight of the rat. At the age and weight range normally used response is maximal at ten times control incorporation, 4h after the administration of 0.3mug or more of oestradiol-17beta. Under these conditions the stimulation of incorporation into the acid-soluble fraction is only 2-2.5-fold. When the purified RNA is separated on polyacrylamide gels the major increase in incorporation of radioactive precursor is found in rRNA and 4S RNA; the formation of the former has been followed from the 45S precursor. Preceding these events by at least 30min, however, is an increase in the incorporation of precursor into RNA species of very high molecular weight, which remained in the first few slices of the gel. The possible significance of these findings is discussed. The increased synthesis of rRNA in response to oestradiol-17beta is more strongly inhibited by actinomycin D than the synthesis of other RNA species. Cycloheximide, depending on time of administration and dosage, inhibits either RNA synthesis or the maturation of rRNA.     

Mechanism of action of silibinin. V. Effect of silibinin on the synthesis of ribosomal RNA, mRNA and tRNA in rat liver in vivo

The influence of the flavonolignane Silibinin on the rate of RNA synthesis in rat livers was studied in detail and the time course of the stimulatory effect was determined: 8 h after i.p. application a maximal increase of about 60% in nuclear RNA synthesis can be observed. The analysis of the RNA by electrophoresis on agarose and by sucrose gradient centrifugation demonstrated that in particular the ribosomal RNA (28S, 18S, 5.8S) synthesis is accelerated followed by enhanced incorporation of rRNA into mature ribosomes. During stimulation also changes in the pattern of 45S RNA can be observed. The synthesis of mRNAs, 5S RNA and tRNAs is not influenced by Silibinin, which was shown after separation of these moieties on oligo(dT)-cellulose, and by polyacrylamid electrophoresis, respectively. The clinically observed enhancement of liver cell regeneration during Silibinin treatment thus can be explained by an increase of the protein synthetic apparatus.     

NEWLY SYNTHESIZED RNA IN NUCLEI ISOLATED FROM NERVE AND GLIAL CELLS

—Nuclear RNA from neurones, astrocytes and other glial cells was pulse-labelled in vivo with [³H]uridine and analysed by sucrose density-gradient centrifugation after various periods of incorporation. Thirty min after the injection of the isotope, rapidly-labelled RNA appeared in all three cell types, a heterogeneous fraction sedimenting above 30S, the others at 25 and 12S. The transformation rate of the two latter components was equally rapid in all three types of nuclei studied. These components are assumed to be of messenger nature. The heavy fractions underwent transformations which in other cells have been described to lead to rRNA formation. The temporal pattern as well as the sequence of changes were similar in nuclei from neurones and astrocytes, the only difference being that a 35S intermediate was found in the former and a 32S in the latter. In non-astrocytic glial nuclei, synthesis and transformation of the 45S component were delayed as compared to the other cell types and the processing of this component may involve both a 32S and a 35S intermediate. Moreover, the radioactivity incorporated in all the nuclear RNA species was always lower in these cells.     

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.     

Changes in the synthesis of ribosomal ribonucleic acid and of poly(A)-containing ribonucleic acid during the differentiation of intestinal epithelial cells in the rat and in the chick.

Epithelial cells were isolated from rat and chick small intestine by techniques which separated subpopulations of differentiating villus and upper crypt cells from each other and from populations of mitotically dividing lower crypt cells. Incorporation of precursors into epithelial-cell DNA, cytoplasmic rRNA and cytoplasmic poly(A)-containing RNA occurred in the lower crypt cells in vivo when precursor was supplied from the vascular system of the intestine. Incorporation of precursor into 28S and 18S rRNA continued in the upper crypt cells, but occurred to only a very slight extent (if at all) in villus cells, whereas incorporation into poly(A)-containing RNA continued (at a diminishing rate) as the differentiating cells migrated along the villi. When precursor was supplied from the intestinal lumen, its incorporation into DNA and into rRNA of crypt cells was not very different from that observed with the other mode of precursor administration, but incorporation into villus-cell poly(A)-containing RNA then occurred at essentially the same rate in all intestinal epithelial cells in vivo. Cytoplasmic poly(A)-containing RNA appeared to turn over in rat crypt cells with a half-life not exceeding 24 h; crypt-cell rRNA showed no turnover and no evidence could be found for the presence of 'metabolic DNA'.     

STUDY OF RNA IN SUBCELLULAR FRACTIONS FROM RAT BRAIN: SIMULTANEOUS INCORPORATION OF [14C]URIDINE AND [3H]METHYL-l-METHIONINE

Abstract— By using a combination of subcutaneous and intraventricular injections of [¹⁴C]uridine and [³H]methyl- l -methionine we have obtained maximum incorporation in about 40 min of both radioactive precursors into nuclear RNA from rat brain. In this nuclear fraction we found at least two different types of RNA that were rapidly labelled. One of them incorporated both [¹⁴C]uridine and [³H]methyl groups and seemed to correspond to species of rRNA and their precursors. The other RNA fraction was less methylated or non-methylated and exhibited sedimentation coefficients distributed along a continuous 8–30 % sucrose density gradient. At least part of the latter type of RNA very probably was mRNA, but much of it must conespond to a different RNA similar to that recently described in HeLa cells by P enman , V esco and P enman (1968).
We also found that labelled 185 and 285 rRNA components began leaving the nucleus for the cytoplasm within 24 to 33 min after the radioactive precursors had been injected, and, in the cytoplasmic fraction, the patterns of incorporation for [¹⁴C]uridine and [³H]-methyl groups were similar for the 18S and 28S rRNA components. We estimate that in this fraction of rat brain the 18S rRNA component was 1·4 times more methylated than the 28S component. We also detected a lower sedimentation coefficient for the non- or slightly methylated, species of soluble RNA found in the cytoplasmic fraction.     

Synthesis and maturation of ribosomal ribonucleic acids in isolated HeLa cell nuclei. A tracer study on the topology of the 45S precursor of ribosomal ribonucleic acids

The synthesis and processing of RNA by isolated HeLa cell nuclei was studied at low ionic strength in the presence of alpha-amanitin. The RNA polymerase reaction, with endogenous template and enzyme, rapidly reaches a plateau dependent on the amount of nuclei. Evidence is presented that incorporation of [(3)H]UMP proceeds only in growing RNA chains, whereas initiation of new RNA chains is arrested. The product formed contains all the main components of the 45S pre-rRNA (precursor of rRNA) maturation pathway (45S, 32S and 20S pre-rRNA; 28S and 18S rRNA). Most of the labelled material is in the mature rRNA components and their immediate precursors, even at very short times of incubation (2min). Small, but definite, 5S and 4S RNA peaks are also observed. At shorter incubation times a substantial amount of [(3)H]UMP is incorporated into RNA molecules in the 24S and 10-16S zones. This RNA material is considered to represent the non-conserved segments of 45S pre-rRNA in the process of nucleolytic degradation. A model for the tracer study of the topology of 45S pre-rRNA, on arrest of rRNA initiation, is discussed. The experimental evidence obtained supports the following structure of 45S pre-rRNA: 5'-end-28S rRNA unit-18S rRNA unit-nonconserved segment-3'-end.     

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.     

THE DIRECT MEASUREMENT OF THE AXOPLASMIC TRANSPORT OF INDIVIDUAL RNA SPECIES: TRANSFER BUT NOT RIBOSOMAL RNA IS TRANSPORTED

Analysis of chick retinal and tectal RNA revealed that in addition to the major cytoplasmic RNAs (rRNA and tRNA), a number of the small mol wt nuclear RNAs (snRNAs) can also be detected. Subfractionation data indicated that one of these molecules, DD′, is of at least 95% nuclear location within the retina. Thus, very little, if any, of the retinal DD′ is available for axoplasmic transport from the retina into the optic nerve and tectum. Following intraocular injection of [³H]uridine, considerable incorporation of isotope into DD′ was observed within the optic tectum after 4, 8 and 16 days. This result indicates the presence of considerable local (i.e. tectal) synthesis. The specific activities of 29S, 18S and 5S rRNA and 4s tRNA relative to that of DD′ were measured in the optic tectum 8 and 16 days after the intraocular introduction of [³H]uridine. The same measurements were also made in intracranially injected animals. While the 29S/DD′, 18S/DD′ and 5S/DD′ specific activity ratios obtained were independent of the injection route, the 4S/DD′ ratio obtained from intraocularly injected animals was significantly greater (at least 2-fold) than that obtained from intracranially injected animals. Similar analysis was also performed with the optic nerve complex at 16 days post-injection with identical results. These results demonstrate that tRNA, but not rRNA, is transported from the retina into the optic nerve and tectum in the 2-day-old chicken.     

Ribonucleic acid synthesis in vitro in primary spermatocytes isolated from rat testis

The incorporation of [(3)H]uridine into RNA was studied quantitatively (by incorporation of [(3)H]uridine into acid-precipitable material) and qualitatively (by phenol extraction and electrophoretic separation of RNA in polyacrylamide gels) in preparations enriched in primary spermatocytes, obtained from testes of rats 26 or 32 days old. The rate of incorporation of [(3)H]uridine into RNA of isolated spermatocytes was constant during the first 8h of incubation, after which it decreased, but the decreased rate of incorporation was not reflected in a marked change in electrophoretic profiles of labelled RNA. In isolated spermatocytes, [(3)H]uridine was incorporated mainly into heterogeneous RNA with a low electrophoretic mobility. Most of this RNA was labile, as shown when further RNA synthesis was inhibited with actinomycin D. Spermatocytes in vivo also synthesized heterogeneous RNA with a low electrophoretic mobility. A low rate of incorporation of [(3)H]uridine into rRNA of isolated spermatocytes was observed. The cleavage of 32S precursor rRNA to 28S rRNA was probably retarded in spermatocytes in vitro as well as in vivo. RNA synthesis by preparations enriched in early spermatids or Sertoli cells was qualitatatively different from RNA synthesis by the spermatocyte preparations. It is concluded that isolated primary spermatocytes maintain a specific pattern of RNA synthesis, which resembles RNA synthesis in spermatocytes in vivo. Therefore isolated spermatocytes of the rat can be used for studying the possible regulation of RNA synthesis during the meiotic prophase.     

The dynamics of cytokine d nitric oxide secretion in mice injected with Tityus serrulatus scorpion venom

AIMS: The effects of Tityus serrulatus venom (TSV) were analysed with respect to the susceptibility of four isogenic mouse, the symptoms following injection of venom and the inflammatory mediators in an experimental model of severe envenomation induced in mice. METHODS: The susceptibility was analysed by lethal dose (LD50) determination, including the symptoms observed during envenomating and glucose levels. The detection of cytokines in serum from mice were analysed using enzyme-linked immunosorbent assay, and nitric oxide (NO) was analysed using nitrite determination. RESULTS: The estimated LD50 values were in micrograms per 100 microliters, and the susceptibility of mice to TSV varies with: (a) mouse strain and route of injection (A/J < BALB/c < C57Bl/6 = DBA); (b) mouse strain and sex (A/J female and male < BALB/c female and male); and (c) body weight (all groups of A/J < BALB/c groups). Among the mouse strains studied, BALB/c mice presented moderate sensibility to TSV, with changes in specific signs and serum levels of glucose, several cytokines and NO, when injected intraperitoneally (i.p.) with 1 LD50 of venom. Sweating, salivation and tremor were the specific signs that preceded death. The maximum levels of glucose in sera from mice injected i.p. with 1 LD50 of TSV were observed 60-90 min post-injection. Significant differences were observed in the time-course of cytokine levels, and the venom induced marked elevations of interleukin (IL)-1alpha, IL-1beta, IL-6, IL-10 and interferon gamma (IFN-gamma). The maximum levels of IL-1alpha and IL-1beta were observed 2 h post-injection. The more pronounced levels of IL-6 were observed 4 h post-injection. There was an early increase in IFN-gamma followed by an even higher level after 4 h. IL-10 levels peaked between 6 and 8 h, and this cytokine probably modulates the secretion of IFN-gamma. Tumor necrosis factor release was not detected in BALB/c mice injected with TSV. NO levels attained maximal release after 2 h, following venom injection, while a second peak for NO was at 6 h. CONCLUSIONS: These findings indicate that the susceptibility to the systemic effects of the venom varies among mice of different haplotypes, and that the cytokines such as IL-1, IL-6, IFN-gamma and NO are strongly involved in the pathogenesis caused by this venom and are correlated with the severity of envenomation.     

Ribonucleic Acid Synthesis in Cells Infected with Herpes Simplex Virus: I. Patterns of Ribonucleic Acid Synthesis in Productively Infected Cells

HEp-2 cells were pulse-labeled at different times after infection with herpes simplex virus, and nuclear ribonucleic acid (RNA) and cytoplasmic RNA were examined. The data showed the following: (i) Analysis by acrylamide gel electrophoresis of cytoplasmic RNA of cells infected at high multiplicities [80 to 200 plaque-forming units (PFU)/cell] revealed that ribosomal RNA (rRNA) synthesis falls to less than 10% of control (uninfected cell) values by 5 hr after infection. The synthesis of 4S RNA also declined but not as rapidly, and at its lowest level it was still 20% of control values. At lower multiplicities (20 PFU), the rate of inhibition was slower than at high multiplicities. However, at all multiplicities the rates of inhibition of 18S and 28S rRNA remained identical and higher than that of 4S RNA. (ii) Analysis of nuclear RNA of cells infected at high multiplicities by sucrose density gradient centrifugation showed that the synthesis and methylation of 45S rRNA precursor continued at a reduced but significant rate (ca. 30% of control values) at times after infection when no radioactive uridine was incorporated or could be chased into 28S and 18S rRNA. This indicates that the inhibition of rRNA synthesis after herpesvirus infection is a result of two processes: a decrease in the rate of synthesis of 45S RNA and a decrease in the rate of processing of that 45S RNA that is synthesized. (iii) Hybridization of nuclear and cytoplasmic RNA of infected cells with herpesvirus DNA revealed that a significant proportion of the total viral RNA in the nucleus has a sedimentation coefficient of 50S or greater. The sedimentation coefficient of virus-specific RNA associated with cytoplasmic polyribosomes is smaller with a maximum at 16S to 20S, but there is some rapidly sedimenting RNA (> 28S) here too. (iv) Finally, there was leakage of low-molecular weight (4S) RNA from infected cells, the leakage being approximately three-fold that of uninfected cells by approximately 5 hr after infection.     

Changes in ribosome population and in nucleic acids during breaking of dormancy and development of apple flower buds

Changes in ribosome population, RNA species and DNA composition in flower buds of apples ( Malus pumila Mill. cvs Ralls and White winter pearmain) were investigated during breaking of dormancy and development. After bursting of flower buds, total ribosomes increased approximately 4-fold, and the polyribosomal fraction increased from 66% to 94% of total ribosomes. The newly synthesized ribosomes were identified by incorporation of radioactive precursor. The observed decrease in specific radioactivity of the monoribosomes is caused by the recruitment of monoribo-somes into polyribosomes after breaking of dormancy.
In both cultivars, the 25S and 18S rRNA peaks increased to a high level on April 8. The peaks of low molecular weight RNA were apparently increased after initial swelling of the flower buds. The DNA of flower buds was separated into three bands by electrophoresis. The median band is the main band of nuclear DNAs. The ahead band and the slow-moving band are satellite components of nuclear DNAs, and they obviously rose after initial swelling of the flower buds. On April 8, when the flower buds had opened, two other small DNA bands could be detected. These results suggest that the changes in level of different ribosome populations, RNA species and DNA composition are related to dormancy breaking development of apple flower buds.