Size of poly (A) +-mRNA in Saccharomyces cerevisiae polysomes.

The size of poly (A) +-mRNA in different classes of yeast polysomes is estimated. The average molecular weight of long-term labelled polysomal poly (A) +-mRNA is about 0,65 x 10(6) daltons. Approximately 60% of the poly (A) +-mRNA polynucleotide chains located at the 5' end, are unprotected by ribosomes and degraded by nucleases upon incubation of cell lysates, to yield a population of poly (A) +-mRNA with an average molecular weight of 0,25 x 10(6) daltons.     

Translation of mRNA for glutamate dehydrogenase and spectrophotometric procedure to follow the enzyme biosynthesis.

Heterogeneous poly (A)-mRNA fraction was isolated from rat liver microsomes using phenol-chloroform extraction, millipore filtration and poly (U)-agarose affinity chromatography. Obtained fractions were characterized with respect to their secondary structure and poly (A) content. Isolated poly (A)-mRNA fraction contained high template activity for glutamate dehydrogenase in cell-free systems with microsomes or polysomes. A spectrophotometric procedure to follow enzyme biosynthesis was also developed.     

Accumulation of polyadenylated mRNA during liver regeneration.

Cytoplasmic and polysomal polyadenylated mRNA [poly(A)+-mRNA] increased by 120% prior to the onset of DNA synthesis during the regeneration of rat liver following partial hepatectomy. Despite this large change in cytoplasmic mRNA and an approximately 50% increase in total nuclear RNA, the amount of polyadenylated nuclear RNA increased by only 15--20% during this time. Neither the average size of nuclear or of cytoplasmic polyadenylated mRNA nor the length of their poly(adenylic acid) [poly(A)] tracts changed during liver regeneration. Polysomal poly-(A)+-mRNA increased proportionately more and at a faster rate than rRNA during the first day following partial hepatectomy. Normal livers contained a substantial proportion of cytoplasmic poly(A)+-mRNA not associated with polysomes but this proportion was not altered in 3-h regenerating liver. Thus, in regenerating liver, most preexisting cytoplasmic mRNA does not appear to be recruited into polysomes prior to the substantial increase in the amount of cytoplasmic poly(A)+-mRNA.     

Serotonin receptors induced by exogenous messenger RNA in Xenopus oocytes

When poly(A)+-mRNA, extracted from rat brain, was injected into Xenopus laevis oocytes, it induced the appearance of serotonin receptors in the oocyte membrane. Application of serotonin to injected oocytes elicited, after a long delay, oscillations in membrane current. The equilibrium potential of this current corresponded with the chloride equilibrium potential. It appears that rat brain mRNA encodes the translation of serotonin receptors into the oocyte membrane. The combination of serotonin with these receptors leads to the opening of membrane channels.     

mRNA of Leghemoglobins from Alfalfa Root Nodules

This paper described the methods of isolation, purification, and in vitro translation of alfalfa leghemoglobin mRNA. It was pointed out that the mRNA of alfalfa leghemoglobin accounts for about 1.4% of total RNA, and 24% of poly (A) +-mRNA of alfalfa nodule. This mRNA has 6, 9, 26S value populations with different molecular weights of about 0.15×106, 0.28×106, 0.48×l06 respectively. These S value mRNAs were coded for different components of leghemoglobin.     

The effect of tryptrophan on nucleocytoplasmic translocation of RNA in rat liver.

The effect of the administration of tryptophan on the transport of nuclear poly (A)-containing mRNA to the cytoplasm in rat liver was investigated. Administration of tryptophan to fasted rats pretreated with cordycepin and actinomycin D led to decreased levels of nuclear poly (A)-mRNA and a concomitant increase in the levels of polyribosomal poly (A)-mRNA in the cytoplasm as determined by measuring in vivo incorporation of labeled precursors into hepatic RNA. Using isolated hepatic nuclei of rats prelabeled in vivo with [14C]orotic acid, there was greater release of labeled poly(A)-mRNA into the incubation medium from nuclei of tryptophan-treated rats than from nuclei of control animals. The increased release of RNA from hepatic nuclei of tryptophan-treated animals was not related to the cell sap present in the media since cell saps from livers of control and experimental rats gave similar results. These results support earlier findings which suggest that in the rat tryptophan increases the rate of translocation of hepatic poly(A)-mRNA from nucleus to cytoplasm.     

Intensified expression of calcium and sodium voltage-operated channels in oocytes ofXenopus following the injection of total RNA from the rat brain

Expression of voltage-gated calcium and sodium ionic channels was found with the help of electrophysiological methods in the membrane of oocytes of theXenopus laevis frog following the injection of total RNA from the brain of 15-to-20-day-old rats. The amplitudes of currents through these channels were much higher than those induced by poly(A)⁺-mRNA from the same source. Barium currents induced by both RNA preparations were insensitive to Bay K 8644 (10 µM) and nitrendipine (50 µM), but were blocked by addition of 100 µM Cd²⁺ to extracellular solution; in both cases -conotoxin GVIA (1 µM) suppressed currents through the expressed calcium channels. The conclusion is that processes responsible for the expression of alien ionic channels in oocyte membrane become more intensive following the injection of total RNA separated in a sucrose gradient than those following the injection of poly(A)⁺-mRNA. The results suggest that the effectiveness of the expression is positively affected by some factors contained in the preparations of total RNA. The question of the nature of these factors remains open.Neirofiziologiya/Neurophysiology, Vol. 25, No. 6, pp. 433–437, November–December, 1993.     

Inhibition of RNA-directed DNA polymerase by aurintricarboxylic acid.

Commercial-grade aurintricarboxylic acid (ATA) inhibits poly(A), poly(C) and viral RNA-directed DNA synthesis by detergent-disrupted virions of Moloney murine leukemia virus. Paper chromatography of crude ATA yields two active components, which appear to behave identically, and at least two inactive components. The concentration of ATA needed to inhibit polymerase activity is proportional to the concentration of viral protein. The inhibition is neither attributable to contaminating heavy metal ions in the ATA preparation nor to chelation by ATA of Mn2+ or Zn2+, the necessary co-factors. Inhibition of the polymerase reaction by ATA greatly increases the Km for the primer [oligo(T)/oligo(dG)], while it only slightly lowers the Vmax and does not affect the Km's for the template [poly(A)/poly(C)] or the substrate (TTP/dGTP). Thus, ATA seems to reduce specifically the affinity of the polymerase for the DNA primer molecule.     

Comparison of the crystallin mRNA populations from rat, calf and duck lens. Evidence for a longer alpha A2-mRNA and two distinct alpha B2-mRNAs in the birds

Total cytoplasmic poly(A)-containing RNA from rat, calf and duck lens was fractionated by electrophoresis in methylmercury hydroxide-containing agarose gels. RNA electrophoresed in parallel lanes was either transferred onto nitrocellulose and hybridized with total cDNA synthesized on the initial mRNA or was recovered from individual gel fractions for in vitro translation in a reticulocyte cell-free system. This allowed the identification and size-characterization of individual mRNA species encoding alpha-, beta-, gamma- and delta-crystallin polypeptides. The 14 S mRNA fraction of rat lens comprises two alpha A2-mRNAs of approximately 1250 and 1350 nucleotides and the alpha AIns-mRNA with a size similar to that of the largest alpha A2-mRNA. The calf lens 14 S mRNA fraction harbors a heterogeneous population of alpha A2-mRNA. In the same fraction another mRNA encoding a polypeptide, designated X, has been found sharing no homology with alpha A sequences. The duck lens alpha A2-mRNA appears to be 400-450 bases longer than the rat and calf lens alpha A2-mRNAs. Furthermore, in contrast to the single alpha B2-mRNA in rat and calf lens, two alpha B2-mRNAs have been identified in duck lens, one, the major species, similar in size to the alpha B2-mRNA in rat and calf lens (800 bases), and the other species 700 nucleotides longer. The large size differences among the alpha A2- and alpha B2-mRNAs most likely reside in their 3'-untranslated sequences.     

Characterization of messenger RNA for chick-embryo DNA polymerase beta and its translation product in vitro

A specific immunoprecipitation method, using rabbit anti-(chick DNA polymerase beta) IgG was applied to detect the polypeptide of DNA polymerase beta among translation products obtained in vitro with mRNA extracted from chick embryos. A polypeptide of Mr = 40 000 was specifically immunoprecipitated from [35S]methionine-labeled translation products and was competitive with the purified DNA polymerase beta for the antibody. Furthermore, the 40 000-Mr translation product obtained in vitro had DNA polymerase activity, which was detected by assay in situ after electrophoresis in a polyacrylamide gel containing DNA. The mRNA for DNA polymerase beta was polyadenylated and its content was estimated as the range of 0.001% of total poly(A)-rich RNA on the basis of [35S]methionine incorporation in the translation in vitro. The size of this mRNA was determined to be about 1800 nucleotides by zone sedimentation and agarose gel electrophoresis under denaturating conditions.     

Isolation and fractionation of total nucleic acids from tissues and cells

A simple and efficient method was devised to permit the isolation of total nucleic acids (poly(A+) and poly(A-) RNAs and DNA) from cells and tissues (e.g. testis) enriched in DNA. Chaotropic reagents (guanidine thiocyanate and LiBr) were utilized to inactivate nucleases rapidly, minimize the viscosity of the homogenization solution and to precipitate RNA selectively (LiBr). Both total and poly(A+)-enriched mRNAs were recovered in a biologically active form as demonstrated by their ability to programme an in vitro translation reaction. High molecular weight DNA (greater than 22 kilobases) was recovered from the LiBr-soluble supernatants by selective ethanol precipitation, subsequently purified and was in a form suitable for further biochemical analysis.     

Radiation-induced mutagenicity and lethality in ames tester strains of salmonella

Mutation and killing induced by X radiation and 60CO gamma radiation were studied in six different histidine-requiring auxotrophs of Salmonella typhimurium. Strain TA100, which is sensitive to base-pair substitutions, and strains TA2637 and TA98, which are sensitive to frameshifts, carry the pKM101 plasmid and exhibit significantly higher radiation-induced mutations compared to their plasmidless parent strains TA1535, TA1537, and TA1538, respectively. Among the plasmid-containing strains, TA98 and TA2637 are much more sensitive to the mutagenic action of radiation than is TA100 based on a comparison with their respective spontaneous mutation rates; however, no uniformity was observed in the responses of the strains to the lethal action of ionizing radiation. The pKM101 plasmid provides partial protection against lethality in TA100 and TA2637, whereas the same plasmid enhances the lethal action of ionizing radiation in TA98. The following conclusions are consistent with these observations: (1) the standard Ames Salmonella assay correctly identifies ionizing radiation as a mutagenic agent; (2) frameshift-sensitive parent strains are more sensitive to the mutagenic effects of ionizing radiation than is the only strain studied that is sensitive to base-pair substitutions; and (3) enhancement of mutagenesis and survival is related to plasmid-mediated repair of DNA damage induced by ionizing radiation and does not involve damage induced by Cerenkov-generated uv radiation which is negligible for our irradiation conditions.     

The role of gibberellin in regulation of dwarf plants development

The effect of exogenously applied gibberellic (GA₃) acid on developmental processes in dwarf pea and dwarf maize seedlings was studied. Plants responded to the phytohormone by accelerated longitudinal growth rate and apparent shortening of developmental phases. Poly(A)-mRNA population isolated from gibberellin-treated pea or maize seedlings exhibited much higher translational activity per mRNA unit in the cell-free wheat germ system when compared with control, untreated plants. Analysis of in vitro translation products made by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS—PAGE) followed by autoradiography and densitometry revealed markedly increased overall intensity of the labelled polypeptide bands in addition to the new protein bands which started to appear in gibberellin-treated pea and maize seedlings while were still not detectable in the control plants of the same age. The banding pattern of translation products programmed by poly(A)-mRNA extracted from 2 days older untreated pea plants resembled that of the gibberellin-treated 2 days younger seedlings. It is concluded that gibberellic acid applied to dwarfs accelerates not exclusively the longitudinal growth of plants but also promotes their transition to the next developmental phases.     

Increased synthesis of ribonucleic acids in mouse liver after treatment with a nonionic detergent.

A single injection of Tween 40 (polyoxyethylene(20)sorbitan monohexanoate) in the dose range of 600--800 mg/kg body weight induced an increased short-term labeling especially of poly(A)-containing mRNAs in mouse liver (using either [3H]orotic acid or [32P]orthophosphate as RNA precursors), and apparently increased the turnover rates of both rRNAs and mRNAs in this organ over a period of 24 h. In the early period (4 h) after the injection of Tween 40, there was also a significant increase in the content of 32P-labeled adenylic acid in microsomal poly(A)-mRNA fraction. The activity of DNA-dependent RNA polymerases in the nuclei of treated animals was stimulated up to 60% over that in control nuclei in the same period after detergent injection.     

Gastroprotective effect of Terminalia arjuna bark on diclofenac sodium induced gastric ulcer

AIM: The present study was aimed to evaluate the effect of methanolic extract of Terminalia arjuna (TA) on diclofenac sodium induced gastric ulcer in experimental rats. METHODS: Animals were induced for gastric ulcer with diclofenac sodium (DIC) (80mg/kg bodyweight in water, orally) and treated orally with TA in various doses ranging from 100mg/kg bodyweight to 500mg/kg bodyweight. The effective dose was 400mg/kg bodyweight, since this dose elicited a maximum reduction in lesion index. The gastroprotective effect of TA was assessed from volume of gastric juice, pH, free and total acidity, pepsin concentration, acid output in gastric juice, the levels of non-protein sulfhydryls (NP-SH), lipid peroxide (LPO), reduced glutathione (GSH), and activities of enzymic antioxidants--super oxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and myeloperoxidase (MPO) in gastric mucosa. The levels of DNA, protein bound carbohydrate complexes--hexose, hexoseamine, sialic acid, fucose in gastric mucosa and gastric juice and the levels of RNA in gastric mucosa were assessed. The stomach tissues were used for adherent mucus content and also for the histological examination. RESULTS: A significant reduction in lesion index was observed in ulcer induced animals treated with TA (DIC+TA) compared to ulcerated rats (DIC). A significant increase was observed in pH, NP-SH, GSH, enzymic antioxidants, protein bound carbohydrate complexes, adherent mucus content, nucleic acids with a significant decrease in volume of gastric juice, free and total acidity, pepsin concentration, acid output, LPO levels and MPO activities in DIC+TA rats compared to DIC rats. Histological studies confirmed the gastroprotective activity of TA. CONCLUSION: From the data presented in this study it could be concluded that T. arjuna acts as an gastroprotective agent probably due to its free radical scavenging activity and cytoprotective nature.     

Genotoxicity of stannous chloride in yeast and bacteria

Stannous chloride was found genotoxic in microbial test systems of the yeast Saccharomyces cerevisiae, in one strain of Salmonella typhimurium and in the Mutoxitest of Escherichia coli. Five isogenic haploid yeast strains differing only in a particular repair-deficiency had the following ranking in Sn2+ -sensitivity: rad52delta>rad6delta>rad2delta>rad4delta>RAD, indicating a higher relevance of recombinogenic repair mechanisms than nucleotide excision in repair of Sn2+ -induced DNA damage. Sn2+ -treated cells formed aggregates that lead to gross overestimation of toxicity when not undone before diluting and plating. Reliable inactivation assays at exposure doses of 25-75 mM SnCl2 were achieved by de-clumping with either EDTA- or phosphate buffer. Sn2+ -induced reversion of the yeast his1-798, his1-208 and lys1-1 mutant alleles, in diploid and haploid cells, respectively, and putative frameshift mutagenesis (reversion of the hom3-10 allele) was observed. In diploid yeast, SnCl2 induced intra-genic mitotic recombination while inter-genic (reciprocal) recombination was very weak and not significant. Yeast cells of exponentially growing cultures were killed to about the same extend at 0.1% of SnCl2 than respective cells in stationary phase, suggesting a major involvement of physiological parameters of post-diauxic shift oxidative stress resistance in enhanced Sn2+ -tolerance. Superoxide dismutases, but not catalase, protected against SnCl2-induced reactive oxygen species as sod1delta had a three-fold higher sensitivity than the WT while the sod2delta mutant was only slightly more sensitive but conferred significant sensitivity increase in a sod1delta sod2delta double mutant. In the Salmonella reversion assay, SnCl2 did not induce mutations in strains TA97, TA98 or TA100, while a positive response was seen in strain TA102. SnCl2 induced a two-fold increase in mutation in the Mutoxitest strain IC203 (uvrA oxyR), but was less mutagenic in strain IC188 (uvrA). We propose that the mutagenicity of SnCl2 in yeast and bacteria occurs via error-prone repair of DNA damage that is produced by reactive oxygen species.