A comparison of sequence complexity of nuclear and polysomal poly(A)+ RNA from different developmental stages ofXenopus laevis

Summary Nuclear poly(A)⁺ and polysomal poly(A)⁺ RNA were isolated from gastrula and early tadpole stages of the amphibianXenopus laevis. Complementary DNA was synthesized from all RNA preparations. Hybridization reactions revealed that at least all abundant and probably most of the less frequent nuclear and polysomal poly(A)⁺ RNA species present at the gastrula stage are also present at the early tadpole stage. On the other hand, there are nuclear RNA sequences at the latter stage which appear, if at all, only at lower concentrations at the gastrula stage. The polysomal poly(A)⁺ RNA hybridization reactions suggest the existence of polysomal poly(A)⁺ RNA sequences at early tadpole stages which are not present in the corresponding gastrula stage RNA.By cDNA hybridization with poly(A)^– RNA it could be shown that most of the poly(A)⁺ containing RNA sequences transcribed into cDNA were also present within the poly(A)^– RNA. It was estimated, that these sequences are 10 fold more abundant within the poly(A)^– polysomal RNA and 3–6 more abundant within the poly(A)^– nuclear RNA as compared to the poly(A)⁺ RNAs.     

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

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

Cytokinins modulate the expression of genes encoding the protein of the light-harvesting chlorophyll a/b complex

Summary Tobacco cell suspension cultures responded to cytokinins (for instance kinetin) by full chloroplast differentiation. The hormone had the effect of stimulating the appearance of a few prominent plastid proteins. Synthesis of the light-harvesting chlorophyl a/b-binding protein (LHCP) in response to kinetin was noteworthy (Axelos M. et al.: Plant Sci Lett 33:201–212, 1984).Poly(A)⁺RNAs were prepared from cells grown in the presence of or without added kinetin. Poly(A)⁺RNA recovery and translation activity were not quantitatively altered by the hormone treatment. In vitro translation of polyadenylated mRNA into precursor polypeptides of LHCP (pLHCP) was quantified by immunoprecipitation and SDS-PAGE fractionation of pLHCP immunoprecipitates: pLHCP-mRNA translating activity was found to be stimulated in parallel to mature LHCP accumulation by kinetin-induced cells.Dot-blot and northern-blot hybridizations of poly(A)⁺RNA were carried out, using as a probe a pea LHCP-cDNA clone (Broglie R. et al.: Proc Natl Acad Sci USA 78: 7304–7308, 1981). A ten-fold increase of the level of pLHCP-encoding sequences was observed in poly(A)⁺RNA prepared from 9-d kinetin-stimulated cells, compared to control cells. Oligo(dT)-cellulose-excluded RNA fractions exhibited very low hybridization levels, in the same ratios as those obtained with poly(A)⁺RNA.Thus, the expression of LHCP-gene activity, in response to kinetin addition to tobacco cell suspension cultures, is regulated by the level of pLHCP-encoding mRNA rather than by translational or post-translational controls. re]19850218 rv]19850605 ac]19850613     

Poly(A) Polymerase from Vaccinia Virus-Infected Cells II. Product and Primer Characterization

The product of the in vitro reaction of a vaccinia virus-induced poly(A) polymerase (see preceding paper) with ATP is shown to be poly(A) by nuclease resistance and by annealing with poly(U). Polyacrylamide gel electrophoresis indicates that the in vitro synthesized poly(A) is associated with large RNA which is sensitive to RNase. RNA which co-purifies with the virus-induced enzyme is similar to vaccinia virus-specific RNA with respect to size and poly(A) content. Double labeling studies indicate that the RNA which co-purifies with the enzyme becomes associated with the poly(A) synthesized in vitro. The poly(A) formed in vitro is located on the 3′-OH terminus of this RNA. During in vitro poly(A) synthesis ³²P from α-[³²P]ATP is transferred to nucleosides other than 2′,3′-AMP, primarily to CMP. Inclusion of poly(U) in the in vitro reactions results in an increase in the transfer of ³²P to UMP.     

Nuclease Stn α from <Emphasis Type="Italic">Streptomyces thermonitrificans</Emphasis>: Characterization of the Associated Adenylic Acid Preferential Ribonuclease Activity

Nuclease Stn α from Streptomyces thermonitrificans hydrolyses DNA and RNA at the rate of approximately 10:l. The optimum pH and temperature for RNA hydrolysis were 7.0 and 45°C. The RNase activity of nuclease Stn α had neither an obligate requirement of metal ions nor was it activated in the presence of metal ions. The enzyme was inhibited by Zn²⁺, Mg²⁺, Co²⁺, and Ca²⁺; inorganic phosphate; pyrophosphate; NaCl; KCl; and metal chelators. It was stable at high concentrations of urea but susceptible to low concentrations of Sodium dodecyl sulfate and guanidine hydrochloride. The rates by which nuclease Stn α hydrolysed polyribonucleotides occurs in the order of poly A >> RNA >> poly U > poly G > poly C. The enzyme cleaved RNA to 3′ mononucleotides with preferential liberation of 3′AMP, indicating it to be an adenylic acid preferential endonuclease.     

Spectroscopic analysis of DNA base-pair opening by Escherichia coli RNA polymerase. Temperature and ionic strength effects

The interaction of Escherichia coli RNA polymerase with poly[d(A-T)] and poly[d-(I-C)] was studied by difference absorption spectroscopy at temperatures, from 5 to 45°C in the absence and presence of Mg²⁺. The effect of KCl concentration, at a fixed temperature, was studied from 12.5 to 400 mM. Difference absorption experiments permitted calculation of the extent of DNA opening induced by RNA polymerase and estimation of the equilibrium constant associated with the isomerization from a closed to an open RNA polymerase-DNA complex. ΔH⁰ and ΔS⁰ for the closed-to-open transition with poly[d(A-T)] or poly[d(I-C)] complexed with RNA polymerase are significantly lower than the values associated with the helix-to-coil transition for the free polynucleotides. For the RNA polymerase complexes with poly[d(A-T)] and poly[d(I-C)] in 50 mM KCl, ΔH⁰ ≈ 15–16 kcal/mol (63–67 kJ/mol) and ΔS⁰ ≈ 50–57 cal/K per mol (209–239 J/K per mol). The presence of Mg²⁺ does not change these parameters appreciably for the RNA polymerase-poly[d(A-T)] complex, but for the RNA polymerase-poly[d(I-C)] complex in the presence of Mg²⁺, the ΔH⁰ and ΔS⁰ values are larger and temperature-dependent, with ΔH⁰ ≈ 22 kcal/mol (92 kJ/mol) and ΔS⁰ ≈ 72 cal/K per mol (approx. 300 J/K per mol) at 25°C, and ΔC_p⁰ 2 kcal/K per mol (approx. 8.3 kJ/K per mol). The circular dichroism (CD) changes observed for helix opening induced by RNA polymerase are qualitatively consistent with the thermally induced changes observed for the free polynucleotides, supporting the difference absorption method. The salt-dependent studies indicate that two monovalent cations are released upon helix opening. For poly[d(A-T)], the temperature-dependence of enzyme activity correlates well with the helix opening, implying this step to be the rate-determining step. In the case of poly[d(I-C)], the same is not true, and so the rate-determining step must be a process subsequent to helix opening.     

Messenger RNA for isocitrate lyase from Chlorella fusca var. vacuolata

Chlorella fusca cultures growing in the light and adapting to acetate in the dark were labelled with adenine-³H and adenine-¹⁴C, respectively. Poly(A)-containing RNA from the mixed cultures was analysed for ¹⁴C/³H ratio after polyacrylamide gel electrophoresis in 98% formamide. The RNA from acetateadapting C. fusca cells contained excess label migrating in the gels at a position equivalent to about 0.85×10⁶ mol.wt. Partially purified anti-isocitrate lyase serum linked to p-aminobenzoyl-cellulose bound 3.5–13% of polysomes from acetate-adapting C. fusca, containing 5–10% of polysomal poly(A)-containing RNA. The antibody-bound poly(A)-containing RNA fraction showed a unimodal size distribution with a mean size of about 0.85×10⁶ mol.wt. after electrophoresis on 4% polyacrylamide gels in 98% formamide. Cell-free translation assays showed a three-fold enrichment of isocitrate lyase mRNA after antibody selection of polysomes and indicated that isocitrate lyase mRNA was abundant in acetate-adapting C. fusca cells.Abbreviations A ₂₆₀ unit The amount of material in 1.0 ml giving an absorbance of 1.0 at 260 nm in a 1 cm light path - PAB-cellulose p-aminobenzoyl-cellulose - SDS sodium lauryl sulphate To whom offprint requests are to be sent     

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

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

Spatial distribution of mRNA during pre-fertilization ovule development in Capsella bursa-pastoris

Summary In situ hybridization of sections of the ovary and ovule of Capsella bursa-pastoris with ³H-polyuridylic acid [³H-poly(U)] showed the presence of polyadenylic acid-containing RNA [poly(A) + RNA] in the cells of the placenta and nucellus. During megasporogenesis there was a decrease in ³H-poly(U) binding activity of the nucellar cells concomitant with the appearance of poly(A) + RNA in the integuments. As the typical eight-nucleate embryo sac was formed, ³H-poly(U) binding was not apparent around the quartet of nuclei at the chalazal end, while it persisted at the micropylar end. Both the egg and synergids as well as the chalazal proliferating tissue showed high concentrations of poly(A) + RNA in their cytoplasm. The results suggest a role for transient localizations of poly(A) + RNA during female sporogenesis and gametogenesis in C. bursa-pastoris.     

Direct demonstration of a complementarity between mRNA and double-stranded sequences of pre-mRNA

The total poly(A)-containing mRNA from mouse liver or Ehrlich ascites carcinoma cells was annealed with denatured ds RNA prepared from heavy nuclear ³H-labeled pre-mRNA of the same tissue. The hybrids formed were detected by binding of complexes to poly(U)-Sepharose columns through the poly(A) of mRNA. With this technique, about 30% of labeled ds RNA was bound to poly(U)-Sepharose after annealing it with an mRNA excess. The proportion of hybrid material detected by RNase treatment was two to three times lower than that obtained by poly(U)-Sepharose binding. The length of the RNase-stable acid precipitable hybrid material consisted of heterogeneous sequences of 10–100 nucleotides long when cytoplasmic, and 10–60 nucleotides long when polysomal mRNA was used in the hybridization reaction. The results obtained show that at least some of the mRNA molecules contain sequences complementary to one of the branches of the pre-mRNA hairpins. These results are compatible with the idea that the hairpin-like sequences in pre-mRNA are localized between mRNA and the non-informative part of the precursor molecule.     

Polyribosomes and messenger RNA from RAT liver mitochondria

Summary Rat liver mitochondrial polyribosomes were isolated free from cytoplasmic ribonucleoprotein contaminations in a number of criteria (sedimentation and buoyant density patterns, ribosomal RNA composition). Heterogeneous poly A containing RNA from mitochondrial polysomes was purified by two-stage cellulose chromatography. This RNA was in vitro labelled with¹²⁵I up to specific activity ~10⁶–10⁷ cts.min^–1.µg ^–1 and used for hybridization experiments with separate complementary strands of mitochondrial DNA and nuclear DNA fragments. The proportions of mitochondrial poly A containing RNA that is complementary to heavy and light strands of mtDNA were respectively 31.5% and 8.3%. Besides, a significant RNA fraction was complementary to unique sequences of nuclear DNA (2–3 copies per haploid genome). The hybrids that were formed possessed a high T_m indicative of a perfect base pairing. A dual intracellular origin of mitochondrial messenger RNA is discussed.     

Endonucleolytic cleavage of murine leukemia virus 35S RNA by microsome-associated nuclease.

Moloney murine leukemia virus 35S RNA (molecular weight 3 to 3.4 × 10⁶) is cleaved by nuclease activity present in microsomal fractions from MLV infected or uninfected mouse embryo cells to two RNA species of approximate molecular weights 1.8 × 10⁶ and 1.5 × 10⁶. Microsomal fractions from MLV infected and uninfected cells also contained nucleolytic activity that solubilized [³H]poly(A)·poly(U) but not [³H]poly(C) or [³H]poly(U); the cleavage of poly(A)·poly(U) was inhibited by ethidium bromide. The cleavage of MLV RNA was also inhibited by ethidium bromide, suggesting double stranded regions in 35S RNA as the site of cleavage.     

Removal of bisphenol A by polymerization and precipitation method using Coprinus cinereus peroxidase

Bisphenol A was efficiently removed by the polymerization and precipitation method using Coprinus cinereus peroxidase. The removal efficiency was optimal between pH 9–10 and at 40 °C with a molar ratio of H₂O₂ to bisphenol A of about 2. To remove 100 mg bisphenol A l^–1, peroxidase was required 5 U ml^–1 at pH 7 and 25 °C and 3 U ml^–1 at pH 10 and 40 °C.     

Cytoplasmic processing events in the polyadenylate region of Physarum messenger RNA

Cytoplasmic processing events in the poly(A) region of mRNA fromPhysarum polycephalum are reviewed. Two classes of poly-containing RNA [poly(A)⁺ RNA] exist in the cytoplasm. One contains very short poly(A) sequences, averaging about 15 adenylate residues, while the other contains relatively long poly(A) sequences, averaging about 60 residues. Molecules with short poly(A) sequences are found exclusively in the polysomes while those with long poly(A) sequences are restricted to the free cytoplasmic mRNP. Since proteins are associated with only the long poly(A) sequences the poly(A) · protein complex is also restricted to the free mRNP. The long poly(A) sequences are relatively short-lived. They are degraded by two distinct processes, a shortening process in which 15–20 residues are gradually removed and a turnover process in which long poly(A) tracts are rapidly converted to the short sequences. This process, along with the dissociation of the poly(A) · protein complex, occurs when poly(A)⁺ RNA molecules located in free mRNP are transferred to the polysomes. Poly(A) · protein complex dissociation appears to preceed poly(A) turnover during translational selection. The significance of these processing events in relation to mRNA maturation is discussed.     

Novel lignocellulolytic ability of Candida utilis during solid-substrate cultivation on apple pomace

Lignocellulolytic enzymes from conventional and non-conventional yeasts are not commonly studied, and they have never been described for Candida utilis species. After solid-substrate cultivation of C. utilis (CCT 3469) on apple pomace, degradation of cellulose, pectin and lignin fragments was observed. Production of the main lignocellulolytic enzymes by C. utilis was investigated and high activity for pectinase (239 U ml^–1) as well as a significant manganese-dependent peroxidase (19.1 U ml^–1) activity was found. Low cellulase (3.0 U ml^–1) and xylanase (1.2 U ml^–1) activities were also observed suggesting that C. utilis may have lignocellulose degradation ability.     

Studies on the Autolysis of Aspergillus oryzae

Mode of action of crystalline nuclease O obtained from autolyzed Aspergillus oryzae on RNA and synthetic homopolymers was examined. Crystalline nuclease O had no strict base specificity, although the velocity of hydrolysis was poly A > poly U > RNA > poly C. This enzyme did not degrade poly G. Digestion of high molecular weight RNA with an excess of this enzyme produced mono-, di- and trinucleotides with 5′-terminal phosphate. The amount of mono-, di- and trinucleotides was, respectively, 13.6, 70.0 and 16.4% of total degradation products. All the four bases were detected in mononucleotide fraction and 3′-terminals and 5′-terminals of oligonucleotides.     

Expression of the linear DNA plasmid pRS64 in the plant pathogenic fungus Rhizoctonia solani

The plant pathogenic isolate RI-64 of anastomosis group 4 of Rhizoctonia solani possesses three linear DNA plasmids (pRS64-1, -2, and -3). Unique poly(A)^– RNA, 0.5 kb in length and hybridizable with the pRS64 DNAs was found in mycelial cells of the isolate RI-64. The overall homology at the nucleotide level between pRS64-1, -2, and -3, and the cDNA prepared from the poly(A)^– RNA was 100%, 73%, and 84%, respectively. The open reading frames found in pRS64-1, -2, and -3 (ORF1-1, ORF2-1, and ORF3-1) are 68 amino acids long. The amino acids sequence showed no significant homology with known proteins. Extracts from Escherichia coli cells expressing ORF1-1 contain a specific protein of 7 kDa. Antisera raised against the ORF1-1 product obtained from E. coli cells cross-reacted with the specific proteins found in the mycelia. The results indicate that the DNA plasmids found in R. solani contain a sequence that encodes a specific protein which may be involved in determination of plant pathogenicity.     

An Extracellular S1-Type Nuclease of Marine Fungus Penicillium melinii

An extracellular nuclease was purified 165-fold with a specific activity of 41,250 U/mg poly(U) by chromatography with modified chitosan from the culture of marine fungus Penicillium melinii isolated from colonial ascidium collected near Shikotan Island, Sea of Okhotsk, at a depth of 123 m. The purified nuclease is a monomer with the molecular weight of 35 kDa. The enzyme exhibits maximum activity at pH 3.7 for DNA and RNA. The enzyme is stable until 75°C and in the pH range of 2.5–8.0. The enzyme endonucleolytically degrades ssDNA and RNA by 3′–5′ mode to produce 5′-oligonucleotides and 5′-mononucleotides; however, it preferentially degrades poly(U). The enzyme can digest dsDNA in the presence of pregnancy-specific beta-1-glycoprotein-1. The nuclease acts on closed circular double-stranded DNA to produce opened circular DNA and then the linear form DNA by single-strand scission. DNA sequence encoding the marine fungus P. melinii endonuclease revealed homology to S1-type nucleases. The tight correlation found between the extracellular endonuclease activity and the rate of H³-thymidine uptake by actively growing P. melinii cells suggests that this nuclease is required for fulfilling the nucleotide pool of precursors of DNA biosynthesis during the transformation of hyphae into the aerial mycelium and conidia in stressful environmental conditions.     

The effect of N-terminal substitutions on the biological activity of MSH fragments

In order to study the role of N-terminal substitutions of peptide sequences related to the active site of α-melanotropin, [Glp⁵]α-MSH(5–10), [Glp⁵, -Phe⁷]α-MSH(5–10), [Sar⁵, -Phe⁷]α-MSH(5–10), [Nle⁴, -Phe⁷]α-MSH(4–10), [N-carbamoyl]α-MSH(5–10), and formyl and acetyl derivatives of α-MSH(5–10), [Gly⁵]α-MSH(5–10) and [Gly⁵, -Phe⁷]α-MSH(5–10), were synthesized in solution. The N-terminal acylations enhance by 2 to 10 times the melanin-dispersing activity of the unsubstituted sequences. Alkylation of the N-terminus does not change the biological activity of the parent peptide, suggesting the necessity of a carbonyl group for increasing the hormonal effect.     

Fermentative production of P(3HB-co-3HV) from propionic acid by Alcaligenes eutrophus in fed-batch culture with pH-stat continuous substrate feeding method

The feeding of propionic acid for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] by Alcaligenes eutrophus ATCC17697 was optimized using a fed-batch culture system. The concentration of propionic acid was maintained at 3 g l^–1 as growth was inhibited by propionic acid in the broth. A pH-stat substrate feeding system was used in which propionic acid was fed automatically to maintain a pH of the culture broth at 7.0. By feeding a substrate solution containing 20% (w/v) propionic acid, 4.9% (w/v) ammonia water [at a molar ratio of carbon to nitrogen (C/N molar ratio) of 10] in cell growth phase, the concentration of propionic acid in the broth was maintained at 3 g l^–1 giving a specific growth rate of 0.4 h^–1. To promote P(3HB-co-3HV) production, two stage fed-batch culture which consisted of the stage for the cell growth and the stage for the P(3HB-co-3HV) accumulation was carried out. When the substrate solution whose C/N molar ratio was 50 was fed in P(3HB-co-3HV) accumulation phase, the cell concentration and the P(3HB-co-3HV) content in the cells reached 64 g l^–1 and 58% (w/w) in 55.5 h, respectively.