[Alkylation of nucleic acids. I. Comparative study of methylation of ribonucleic acids in aqueous and organic solution].
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Alkylation↗
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Composition of ribonucleic acid from various parts of spider oocytes.
Microphoretic purine-pyrimidine analyses of the ribonucleic acid (RNA) in nucleoli, nucleoplasm, cytoplasm, and yolk nuclei of spider oocytes have been carried out. The material necessary for the analyses was isolated by micromanipulation. Determinations of the amounts of RNA in the different parts of the cell were also performed. No differences between the composition of RNA in the nucleolus and the cytoplasm could be disclosed. Nucleoplasmic RNA was, on the other hand, distinctly different from that in the nucleolus and in the cytoplasm. The difference lies in the content of adenine, which is highest in nucleoplasmic RNA. The few analyses carried out on yolk nuclei showed their RNA to be variable in composition with a tendency to high purine values. The cytoplasm contains about 99 per cent of the total RNA in these cells, the nucleoplasm about 1 per cent, and the nucleolus not more than 0.3 per cent, although the highest concentrations are found in these latter structures. When considered in the light of other recent findings the results are compatible with the view that nucleolar RNA is the precursor of cytoplasmic RNA.
Adenine↗
De novo biosynthesis and base composition of total and ribosomal ribonucleic acids during early development in Artemia sp.
De novo synthesis of total and ribosomal ribonucleic acids has been studied during the early stages of Artemia sp. development. By in vivo incorporation studies of [14C]HCO3- an increase has been found in both total and ribosomal RNA synthesis post hatching, with a similar distribution of radioactivity and base composition.
Animals↗
Interleukin-1 type I receptor messenger ribonucleic acid expression in human endometrium throughout the menstrual cycle.
OBJECTIVE: To investigate the messenger ribonucleic acid (mRNA) expression of interleukin-1 (IL-1) type I receptor in the endometrial tissue of normal patients during the menstrual cycle. DESIGN: Prospective longitudinal study. SETTING: Department of Obstetrics and Gynecology, Stanford University Medical Center, Stanford, California. PATIENTS: Twenty fertile women between 19 and 41 years of age underwent hysterectomy for benign reasons (n = 9) and laparoscopy for tubal ligation (n = 11). In all cases, endometriosis was not visualized. INTERVENTIONS: Endometrial biopsy using the Novak curette was obtained at the time of surgery. MAIN OUTCOME MEASURE: Total RNA extracted from unfractioned endometrial tissue was analyzed on Northern blots by using specific complementary deoxyribonucleic acid probes. RESULTS: We found IL-1 type I receptor mRNA expression in endometrial tissue throughout the entire menstrual cycle. However, IL-1 type I receptor mRNA levels were significantly higher during both early and late luteal phases than follicular and midluteal phases. CONCLUSIONS: Our results demonstrate the presence of the IL-1 system in the human endometrium and that the receptor is regulated throughout the menstrual cycle with a 4.1-fold increased expression of the IL-1 receptor gene in the early luteal phase compared with preovulatory endometrium.
Adult↗
Quantitation of ribonucleic acids using 18O labeling and mass spectrometry.
A previous limitation in the analysis of ribonucleic acids (RNAs) by mass spectrometry (MS) has been the inability to obtain quantitative information relating to total RNA, RNA subunits, and undermodified nucleosides in a straightforward manner. Here, a simple and rapid method has been developed for the relative quantitation of small RNAs using 18O labeling and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). One RNA sample is digested with RNase T1 in 18O-labeled ("heavy") water with the 18O being incorporated at the 3'-phosphate end of oligonucleotides upon hydrolysis. A second RNA sample is digested with RNase T1 in normal ("light") water. The two samples are then combined and analyzed by MALDI-MS. Relative ion abundances of the light- and heavy-water digestion products, which are separated by 2 Da due to the isotopic mass of 18O, reveal relative quantitation information from the two RNA samples. The accuracy and reproducibility of this approach were tested on 18 known RNA samples and 4 unknown RNA samples. The coefficients of variation for quantitation were found to be generally below 15% when using MALDI-MS. The approach yields accurate quantitative information for heavy-to-light ratios greater than 1:2. This method should prove useful for quantitatively characterizing variations in RNA production and variations in the amount of posttranscriptionally modified nucleosides.
Isotope Labeling↗
Abortive initiation and long ribonucleic acid synthesis.
In vitro transcription assays have been used to study the rate of ribonucleic acid (RNA) synthesis from the Escherichia coli lactose promotor mutant lacL8UV5 contained on a 203-bp (base pair) restriction fragment. The half-life of long (63-base) RNA production from heparin-resistant RNA polymerase-promotor complexes was found to be related to the amount of oligonucleotides released during the initiation process (abortive initiation). Studies indicate that once a ternary complex between the promoter, RNA polymerase, and a newly synthesized RNA seven and nine nucleotides long is formed, abortive initiation is reduced and the rate of synthesis of long RNAs is increased. The promoter for the left inverted repeat of the transposable element Tn5 was also examined. It was observed to have a much slower rate of production of long RNAs, and it released oligonucleotides 4 times as often as the lactose promoter. The correlation between the amount of abortive initiation and the half-time of long RNA production is discussed.
Base Composition↗
Distribution of cytokinin-active nucleosides in isoaccepting transfer ribonucleic acids from Agrobacterium tumefaciens.
The cytokinin-active isoprenoid nucleosides of Agrobacterium tumefaciens transfer ribonucleic acid were identified by high-pressure liquid chromatography, permethylation, and mass spectroscopy. Besides the expected 6-[(3-methylbut-2-enyl)amino]-9-(beta-D-ribofuranosyl)purine (i6A) and its 2-methylthio derivative (ms2i6A), substantial amounts of cis- and trans-ribosylzeatin (io6A) and cis-2-(methylthio)ribosylzeatin (c-ms2io6A) were present. These hydroxylated side chain derivatives are normally characteristic of plant tRNA. Fractionation of the total bacterial tRNA on BD-cellulose and RPC-5 allowed isolation of purified iso-accepting species whose cytokinin nucleoside contents were then determined. Distribution of the isoprenoid nucleosides among the U-group tRNA species was not uniform. cis-Ribosylzeatin was found almost exclusively in one tRNASer while ms2io6A was found predominantly in tRNAPhe, tRNASer, and tRNATyr. Not all cytokinin-active species were found in every member of the U-group tRNAs. The only species present in tRNATrp was i6A; it contained no zeatin derivatives. The hydroxylation and methylthiolation processes appear to be highly specific and dependent upon tRNA structure or sequence.
Amino Acyl-tRNA Synthetases↗
Comparison of ribonucleic acid homopolymer ionization energies and charge injection barriers.
Thin films of guanosine and uridine ribonucleic acid (RNA) homopolymers (poly rG, poly rU) were grown in high vacuum in several steps on highly oriented pyrolytic graphite (HOPG) using electrospray deposition. Between deposition steps, the sample surface was characterized with X-ray and ultraviolet photoemission spectroscopy (XPS, UPS). The resulting spectra series allowed the determination of the orbital alignment at the HOPG interface, as well as the ionization energies of the homopolymer thin films. Comparison with earlier results on cytidine and adenosine RNA homopolymers (poly rC, poly rA) indicates significant ionization energy and charge injection barrier differences between purines and pyrimidines.
Graphite↗
The Influence of Auxin and Ethylene on Chromatin-directed Ribonucleic Acid Synthesis in Soybean Hypocotyl.
Soybean seedlings treated with ethylene exhibited small increases in ribonucleic acid content in the elongating section of the hypocotyl. Chromatin isolated from the elongating section of ethylene-treated seedlings showed a 35 to 60% increase in the capacity for RNA synthesis. The ethylene-induced response was saturated at 1 microliter/liter of ethylene and was fully expressed after 3 hours. Auxin caused marked accumulation of RNA and DNA in the elongating and basal tissue of the hypocotyl. Chromatin isolated from these auxin-treated tissues showed an 8- to 10- fold increase in RNA synthetic capacity as measured in vitro. Ethylene added with auxin reduced the auxin enhancement of nucleic acid synthesis in the elongating and basal tissues. Both ethylene and auxin treatment of the seedlings inhibited nucleic acid accumulation and chromatin activity in the apical tissue. Ethylene did not appear to mediate the auxin effects on nucleic acid synthesis in soybean hypocotyl with the possible exception of inhibition in the apical tissue.The RNA which was synthesized by chromatin isolated from control and auxin- and ethylene-treated tissues was characterized by nearest neighbor analyses. The nearest neighbor frequencies of the RNA products synthesized by chromatin isolated from auxin- and ethylene-treated hypocotyl tissue were different from each other and different from the control RNA product.Seedlings treated in sealed containers exhibited growth, RNA, and DNA responses, especially to ethylene, different from those of seedlings treated in continuous flow containers.
Journal Article↗
Inhibition of ribonucleic acid polymerase by a bacteriocin from Bacteroides fragilis.
The Bacteroides fragilis bacteriocin which inhibits ribonucleic acid (RNA) polymerase activity had a narrow activity spectrum in vivo and only inhibited the growth of certain B. fragilis strains. In vitro the bacteriocin was not specific and inhibited RNA polymerases from widely diverse bacterial genera. RNA polymerases from rifampin-resistant strains of Bacteroides thetaiotaomicron and Clostridium acetobutylicum were resistant to the bacteriocin in vitro. Purified bacteriocin bound to partially purified RNA polymerase, and both proteins were cosedimented in a glycerol gradient. In the RNA polymerase reaction, the bacteriocin acted as a competitive inhibitor for adenosine, cytidine, and uridine 5'-triphosphates and as a noncompetitive inhibitor for guanosine 5'-triphosphate. The bacteriocin did not inhibit RNA polymerase from chicken embryos.
Bacteriocins↗
Measurement of microbial activity and growth in the ocean by rates of stable ribonucleic Acid synthesis.
A relatively simple and extremely sensitive technique for measuring rates of stable ribonucleic acid (RNA) synthesis was devised and applied to bacterial cultures and seawater samples. The procedure is based upon the uptake and incorporation of exogenous radiolabeled adenine into cellular RNA. To calculate absolute rates of synthesis, measurements of the specific radioactivity of the intracellular adenosine 5'-triphosphate pools (precursor to RNA) and of the total amount of radioactivity incorporated into stable cellular RNA per unit time are required. Since the rate of RNA synthesis is positively correlated with growth rate, measurements of RNA synthesis should be extremely useful for estimating and comparing the productivities of microbial assemblages in nature. Adenosine 5'-triphosphate, adenylate energy charge, and rates of stable RNA synthesis have been measured at a station located in the Columbian Basin of the Caribbean Sea. A subsurface peak in RNA synthesis (and therefore growth) was located within the dissolved oxygen minimum zone (450 m), suggesting in situ microbiological utilization of dissolved molecular oxygen. Calculations of the specific rates of RNA synthesis (i.e., RNA synthesis per unit of biomass) revealed that the middepth maximum corresponded to the highest specific rate of growth (420 pmol of adenine incorporated into RNA.day) of all depths sampled, including the euphotic zone. The existence of an intermediate depth zone of active microbial growth may be an important site for nutrient regeneration and may serve as a source of reduced carbon for mesopelagic and deep sea environments.
Journal Article↗
Mycobacterial ribonucleic acid: comparison with mycobacterial cell wall fractions for regression of murine tumor growth.
Mycobacterial ribonucleic acid (RNA) and cell wall skeleton fraction isolated from H37Ra caused P-815 mastocytoma regression in DBA/2 mice provided the animals were presensitized with freshly harvested living H37Ra cells. In the absence of presensitization, only the RNA fraction inhibited. Cell wall skeleton fraction, under these conditions, stimulated tumor growth. Cell wall lipids (from H37Ra) added to H37Ra cell wall skeleton fraction did not increase the inhibitory activity of cell wall skeleton fraction alone. Mycobacterial RNA appeared to be an effective inhibitor of P-815 mastocytoma metastases as shown by (i) the inhibition of a second footpad lesion distant from the one treated and (ii) increase in survival time.
Animals↗
Stable messenger ribonucleic acid and germination of Myxococcus xanthus microcysts.
We have examined germination, protein synthesis and ribonucleic acid (RNA) synthesis by microcysts of the fruiting myxobacterium Myxococcus xanthus. The morphological aspects of microcyst formation were completed at about 2 hr after induction had begun. In such microcysts, germination, RNA synthesis, and protein synthesis were inhibited by actinomycin D (Act D). At 6 hr after induction, germination and protein synthesis had become relatively resistant to Act D, whereas RNA synthesis was inhibited by about 95%. Experiments with (3)H-Act D indicated that the deoxyribonucleic acids of both young and old microcysts bind Act D equally. Resistance of germination to Act D was acquired 4 to 5 hr after induction of microcyst formation, and was due to an Act D-sensitive synthesis at that time. Vegetative cells and microcysts were pulsed with uridine-5-(3)H and chased for 60 min; the RNA was extracted and analyzed by means of sucrose density gradient centrifugation and gel electrophoresis. Both microcysts and vegetative cells were found to contain grossly the same types of RNA in the same proportions. RNA pulse-labeled in microcysts was more stable than that in vegetative cells. No particular portions of the microcyst pulse-labeled RNA were selectively stabilized. These data indicate that a stable messenger RNA required for synthesis of germination proteins was synthesized during microcyst formation. This may be the same as the RNA synthesized 4 to 5 hr after initiation of microcyst formation. We suggest that the existence of such stable messenger RNA in microcysts is consistent with the limited biosynthetic activities of such cells.
Bacteria↗
Crystal formation by a ribonucleic acid polymerase mutant of Bacillus subtilis.
A crystalline inclusion has been observed in a ribonucleic acid polymerase mutant of Bacillus subtilis which is conditionally temperature sensitive only during sporulation. The crystal is formed at the permissive temperature in 1 to 2% of the sporulating (stage III-IV) cells; about 85% of the cells sporulate normally, while the cells with crystals do not sporulate. The wild type does not form crystals at either the permissive (30 C) or the nonpermissive (47 C) temperatures. The crystal may result from altered transcription during sporulation at 30 C.
Bacillus subtilis↗
Precursor of 5S ribosomal ribonucleic acid in the blue-green alga Anacystis nidulans.
The maturation of 5S ribosomal ribonucleic acid (rRNA) in the obligately photoautotrophic unicellular blue-green alga Anacystis nidulans has been studied by using polyacrylamide gel electrophoresis and T1 ribonuclease oligonucleotide analysis. A. nidulans mature 5S rRNA (m5) is of approximately the same molecular weight as the 5S rRNA of Escherichia coli, and is derived by cleavage of a precursor (p5) containing a few (three to six) additional nucleotides. Some of these additional nucleotides occur at the 5' end of the precursor molecule; others may occur at the 3' end. Kinetic experiments indicate that precursors of mature 5S rRNA larger than p5 either do not exist or are very transient in A. nidulans. These results are discussed in relation to those obtained with other prokaryotes.
Autoradiography↗
Enhanced level and metabolic regulation of methionyl-transfer ribonucleic acid synthetase in different strains of Escherichia coli K-12.
The methionyl-transfer ribonucleic acid (tRNA) synthetase of Escherichia coli K-12 eductants carrying P2-mediated deletions in the region of the structural gene of this enzyme was investigated. No structural alteration of this enzyme was observed in three eductants examined. These were isolated from strain AB311, which had a threefold higher level of methionyl-tRNA synthetase than most haploid strains examined. In two of the three eductants studied, the level of this enzyme was twofold higher than in their parental strain regardless of growth conditions used. In contrast, isoleucyl-, leucyl-, and valyl-tRNA synthetases had similar levels in all strains examined. Like valyl-tRNA synthetase, but to a lesser extent, methionyl-tRNA synthetase was subject to metabolic regulation. Coupling between the level of methionyl-tRNA synthetase and growth rate was observed even in strains that had an enhanced level of methionyl-tRNA synthetase. These results suggest that the formation of methionyl-tRNA synthetase remains subject to metabolic regulation even when the repression-like mechanism that controls the synthesis of this enzyme is altered. In addition, we report that in the merodiploid strain EM20031, which was haploid for the valyl-tRNA synthetase structural gene and diploid for the structural genes of methionyl-tRNA synthetase and D-serine deaminase, the levels of these latter two enzymes varied to a minor yet significant extent with the phosphate concentration of the culture medium; under the same conditions, the level of valyl-tRNA synthetase remained unchanged. Moreover, no variation of the levels of these three enzymes in response to phosphate was observed in the haploid strain HfrH. These results indicate that in the merodiploid strain EM20031, which carries the episome F32, the number of episomes per chromosome varies to some extent according to the phosphate concentration of the culture medium.
Amino Acyl-tRNA Synthetases↗
Characterization of the hybridization between purified 16S and 23S ribosomal ribonucleic acid and ribosomal deoxyribonucleic acid from Escherichia coli.
An assay to distinguish specifically between 16S and 23S ribosomal ribonucleic acid (rRNA) has been developed. The assay involves hybridization of radioactive rRNA to deoxyribonucleic acid (DNA) from lambdailv5 transducing phage, which carries an rRNA transcription unit. Radioactive 16S or 23S rRNA can be specifically and completely competed from hybrids by using highly purified nonradioactive 16S or 23S competitor RNA, respectively. The preparation and purification of 16S rRNA and 23S rRNA are described in detail. The hybridization assay is extremely sensitive and efficient; 65 to 70% or more of the input radioactivity hybridizes to specific DNA in the absence of homologous competitor RNA, and at saturation virtually all of the specific DNA sequences are hybridized to rRNA. The results indicate that: (i) the 16S rRNA and 23S rRNA prepared as described are greater than 99% pure, (ii) 16S RRNA and 23S rRNA hybridize with equal efficiency and in equal molar amounts of lambdailv5 DNA; (iii) at saturation, about one molecule of 16S and one molecule of 23S rRNA are hybridized per genome equivalent of lambda ilv 5 DNA; (iv) essentially no cross-hybridization occurs between 16S and 23S rRNA; and (v) the sequence homology between 16S and 23S rRNA is negligible.
Coliphages↗
Rate of synthesis of polyadenylate-containing ribonucleic acid during the yeast cell cycle.
The rate of synthesis of polyadenylate-containing ribonucleic acid is constant throughout the cell cycle of Saccharomyces cerevisiae.
Cell Division↗