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Leucyl-transfer ribonucleic acid synthetase from a wild-type and temperature-sensitive mutant of Salmonella typhimurium.

Leucyl-transfer ribonucleic acid (tRNA) synthetase was purified 100-fold from extracts of Salmonella typhimurium. The partially purified enzyme had the following K(m) values: leucine, 1.1 x 10(-5)m; adenosine triphosphate, 6.5 x 10(-4)m; tRNA(I) (Leu), 4.1 x 10(-8)m; tRNA(II) (Leu), 4.3 x 10(-8)m; tRNA(III) (Leu), 5.3 x 10(-8)m; and tRNA(IV) (Leu), 2.9 x 10(-8)m. The tRNA(Leu) fractions were isolated from Salmonella bulk tRNA by chromatography on reversed-phase columns and benzoylated diethylaminoethyl cellulose. The enzyme had a pH optimum of 8.5 and an activation energy of 10,400 cal per mole, and was inactivated exponentially at 49.5 C with a first-order rate constant of 0.064 min(-1). Strain CV356 (leuS3 leuABCD702 ara-9 gal-205) was isolated as a mutant resistant to dl-4-azaleucine and able to grow at 27 C but not at 37 C. Extracts of strain CV356 had no leucyl-tRNA synthetase activity (charging assay) when assayed at 27 or 37 C. Temperature sensitivity and enzyme deficiency were caused by mutation in the structural gene locus specifying leucyl-tRNA synthetase. A prototrophic derivative of strain CV356 (CV357) excreted branched-chain amino acids and had high pathway-specific enzyme levels when grown at temperatures where its doubling time was near normal. At growth-restricting temperatures, both amino acid excretion and enzyme levels were further elevated. The properties of strain CV357 indicate that there is only a single leucyl-tRNA synthetase in S. typhimurium.

Adenosine Triphosphate↗

Control of stable ribonucleic acid chain initiation in Escherichia coli during diauxie lag.

During glucose-lactose diauxie lag, Escherichia coli synthesizes ribonucleic acid (RNA) at about 7% of the rate in exponentially growing cells. RNA synthesis could be restored to the log-phase rate within 3 min after the addition of glucose to the culture (shift-up). This stimulation occurred equally well in the presence of chloramphenicol and therefore did not require the synthesis of new proteins. RNA synthesis could also be stimulated by the addition of glycerol, but only after a delay of 15 to 20 min. Glycerol stimulation occurred more rapidly in cells preadapted by prior growth on glycerol and occurred immediately in a mutant (glpR) constitutive for the enzymes of glycerol catabolism. Measurement of a parameter related to the rate of initiation of stable (ribosomal and transfer) RNA chains showed that the initiation of transcription of these stable RNA species was completely inhibited during the diauxie lag, but could be restored to the rate characteristic of log-phase cells by shift-up with glucose. Sucrose gradient analysis of RNA purified from cells after a glucose shift-up indicated an increased rate of accumulation of the ribosomal and transfer RNA species. It is proposed that a specific mechanism to inhibit the initiation of transcription of ribosomal and transfer RNA operates during diauxie lag. This inhibitory mechanism is governed by the level of cellular energy resources insofar as the inhibition is readily reversed by metabolizable energy sources.

Carbon Isotopes↗

Dominant and semidominant mutations leading to thermosensitivity of ribonucleic acid biosynthesis in Saccharomyces cerevisiae.

Different dominant thermosensitive mutations affecting the same gene were selected in Saccharomyces cerevisiae. Ribonucleic acid (RNA) synthesis decreased rapidly and markedly at 37 C in all the mutants whether they were in a homozygous or a heterozygous state. Protein biosynthesis was at first unaffected and then decreased slowly, stopping after 5 h. Measurements of RNA biosynthesis in isolated nuclei as well as in vitro activities of RNA polymerases A and B at 22 and 37 C failed to reveal any difference between mutants and the wild type. Analysis of the nature of the residual RNAs synthesized at the high temperature in the mutants showed a small relative increase in the messenger RNA fraction, but it was not sufficient to indicate a specific inactivation of RNA polymerase A activity. The results suggest an impairment in a common regulatory element for all RNA polymerases acting at the level of the initiation of transcription. Similar mutants with a semidominant phenotype were obtained in which the lesions were in two other unlinked loci.

Antifungal Agents↗

Encapsidation of yeast killer double-stranded ribonucleic acids: dependence of M on L.

Virus-like particles containing either L or M double-stranded ribonucleic acid (dsRNA) were isolated from a killer toxin-producing strains of Saccharomyces cerevisiae (K+ R+). At least 95% of M- and 87% of L-dsRNA were recovered in virus-like particle-containing fractions. The major capsid polypeptides (ScV-P1) of both L and M virus-like particles were shown to be identical, and 95% of the cellular ScV-P1 was found in the virus-like particle-containing fractions. Since L-dsRNA encodes ScV-P1, provision of this protein for encapsidation of M-dsRNA defines at least one functional relationship between these dsRNA genomes and associates the L-dsRNA with the killer character. If encapsidation of M-dsRNA is essential for its replication or expression, then L-dsRNA plays an essential role in maintenance or expression of the killer phenotype. The relationship between the L- and M-dsRNA genomes would be analogous to that between a helper and a defective virus. The presence of only minor quantities or uncomplexed dsRNA and ScV-P1 suggests that their production is stringently coupled.

Capsid↗

Synthesis and function of ribonucleic acid polymerase and ribosomes in Escherichia coli B/r after a nutritional shift-up.

The syntheses of stable ribosomal ribonucleic acid (RNA) and transfer RNA in bacteria depend on the concentration and activity of RNA polymerase and on the fraction of active RNA polymerase synthesizing stable RNA. These parameters were measured in Escherichia coli B/r after a nutritional shift-up from succinate-minimal to glucose-amino acids medium and were found to change in complex patterns during a 1- to 2-h period after the shift-up before reaching a final steady-state level characteristic for the postshift growth medium. The combined effect of these changes was an immediate, one-step increase in the exponential rate of stable RNA synthesis and thus of ribosome synthesis. This suggests that the distribution of transcribing RNA polymerase over ribosomal and nonribosomal genes and the polymerase activity are continuously adjusted during postshift growth to some growth-limiting reaction whose rate increases exponentially. It is proposed that this reaction is the production of amino-acylated transfer RNA and that is exponentially increasing rate results in part from a gradually increasing concentration of aminoacyl transfer RNA synthetases after a shift-up. This idea was tested and is supported by a computer simulation of a nutritional shift-up.

Bacterial Proteins↗

Cloning and characterization of the gene for Escherichia coli tryptophanyl-transfer ribonucleic acid synthetase.

From a Clark-Carbon plasmid containing trpS, the structural gene for the tryptophanyl-transfer ribonucleic acid synthetase of Escherichia coli, we subcloned a 2.6-kilobase fragment that has trpS and its neighboring regions. The location and orientation of trpS in the deoxyribonucleic acid insert was determined by deoxyribonucleic acid sequencing. In vitro transcription experiments and S1 nuclease mapping studies with in vivo message established that transcription is initiated at the same site in vivo and in vitro, approximately 58 base pairs upstream from the trpS coding region. We also describe the construction of an inphase trpS-lacZ gene fusion that is under the control of the trpS promoter and encodes a hybrid protein with beta-galactosidase activity.

Amino Acyl-tRNA Synthetases↗

Effects of cycloheximide and 5-fluorouracil on formation of low-molecular-weight ribonucleic acid in yeast.

The effects of cycloheximide and 5-fluorouracil on the formation of low-molecular-weight ribonucleic acid (RNA) in yeast were investigated. Both compounds were found to affect the synthesis of low-molecular-weight RNA of ribosomal origin more than transfer RNA but less than the high-molecular-weight ribosomal RNA. 5-Fluorouracil-containing transfer RNA was separated from normal transfer RNA by chromatography on diethylaminoethyl cellulose at 80 C in the presence of 7 m urea.

Adenine↗

Effect of endotoxin and cortisone on synthesis of ribonucleic acid and protein in livers of mice.

The effect of cortisone and endotoxin, singly and in combination, on ribonucleic acid (RNA) synthesis in livers of adrenalectomized mice was determined. This was accomplished by measuring the incorporation either of inorganic (32)P or of (14)C-orotic acid into the RNA. Under similar conditions, the effect of these agents on the rate of protein synthesis was examined with the use of (14)C-leucine. Bacterial endotoxin was found to augment the uptake of isotope in the RNA and in the protein of the liver. These reactions did not appear to be mediated via the pancreatic hormone insulin, which was found to depress the incorporation of the radioactive compounds into RNA. Cortisone increased the uptake of isotope in liver RNA but depressed the incorporation of leucine into hepatic protein. These results indicate that the previously observed ability of endotoxin to prevent the hormone induction of hepatic enzymes, such as tryptophan oxygenase, is not associated with impaired synthesis of liver RNA or protein.

Adrenalectomy↗

Transcription of the influenza ribonucleic acid genome by a virion polymerase. II. Nature of the in vitro polymerase product.

The properties of the ribonucleic acid (RNA) synthesized by the influenza (WSN) virion polymerase have been investigated. Most of the product RNA is synthesized in association with virion RNA species from which it can be released by heat treatment as single-stranded, ribonuclease-sensitive polynucleotides (average molecular weight, 2-hr sample, about 10(5) daltons). At least 95% of the product is complementary to the viral RNA species. On the basis of the molar ratio of the RNA species isolated from a (3)H-uridine-labeled virus preparation, it was calculated that the WSN genome consists of seven pieces of RNA with a sum molecular weight of about 5 x 10(6) daltons.

Animals↗

Specific demonstration of ribonucleic acid by chemical bromination and immunohistochemistry.

In this report we describe a specific staining procedure for detection of ribonucleic acid (RNA), based on bromination of uracil and subsequent immunohistochemical visualization of 5-bromouracil in RNA. This method is applicable for both cryostat and glycol methacrylate (GMA)-embedded sections. Cryostat sections must be fixed in formaldehyde, whereas tissue pieces to be embedded in GMA are fixed in cold acetone. Before bromination, sections must be treated with trypsin. Bromination was performed in a solution of bromine in potassium bromide. After bromination, excess bromine was removed with sodium bisulfite. The monoclonal antibody MoBu-1 specifically bound to brominated RNA. Ribonuclease digestion, in contrast to deoxyribonuclease digestion, abolished staining. This method makes possible precise localization of RNA, especially well demonstrated in plastic-embedded sections.

Animals↗

Expression of melanin-concentrating hormone receptor messenger ribonucleic acid in tumor tissues of pheochromocytoma, ganglioneuroblastoma, and neuroblastoma.

Expression of melanin-concentrating hormone (MCH) receptor messenger ribonucleic acid (mRNA) was studied by RT-PCR and Northern blot analysis in human brain; pituitary; adrenal glands; tumor tissues of adrenal tumors, ganglioneuroblastomas, and neuroblastomas; and various cultured tumor cell lines. RT-PCR analysis showed that MCH receptor mRNA was widely expressed in brain tissues, pituitary, normal portions of adrenal glands (cortex and medulla), tumor tissues of adrenocortical tumors (12 of 13 cases), pheochromocytoma (all 7 cases), ganglioneuroblastoma (1 case), neuroblastoma (all 5 cases), and various cultured tumor cell lines (6 of 7 cell lines), including 2 neuroblastoma cell lines. Northern blot analysis showed the expression of MCH receptor mRNA ( approximately 2.4 kb) only in the tumor tissues of 5 pheochromocytomas, 1 ganglioneuroblastoma, and 4 neuroblastomas, indicating that the expression levels of MCH receptor mRNA are much higher in these tumors than in the other tissues. These findings raised the possibility that MCH or MCH-like peptides may be related to the pathophysiology of these neural crest-derived tumors.

Adrenal Gland Neoplasms↗

Luteinizing hormone receptor, steroidogenesis acute regulatory protein, and steroidogenic enzyme messenger ribonucleic acids are overexpressed in thecal and granulosa cells from polycystic ovaries.

Recent data suggest that steroidogenic enzyme messenger ribonucleic acids (mRNAs) may be overexpressed in thecal cells, and LH receptors may be prematurely expressed in granulosa cells in women with polycystic ovaries. The purpose of this study was to determine whether there is abnormal gene expression in thecal and granulosa cells from polycystic ovaries. Ovarian tissue specimens were obtained from 12 women with PCOS and 24 regularly cycling control women. The granulosa cells and the theca interna were microdissected from individual follicles. LH receptor, steroidogenesis acute regulatory protein (StAR), cholesterol side-chain cleavage cytochrome P450 (CYP11A), and 17alpha-hydroxylase/C(17-20) lyase cytochrome P450 (CYP17) mRNAs were measured by RT-PCR. There was no difference between 3- to 7-mm control follicles and dominant follicles with respect to LH receptor mRNA expression in either thecal or granulosa cells. CYP11A and CYP17 mRNAs were higher in thecal cells from 3- to 7-mm follicles than in dominant follicles, but StAR expression was not different. In granulosa cells, StAR and CYP11A mRNA expression was higher in dominant follicles than in 3- to 7-mm follicles. The mean levels of LH receptor, StAR, CYP11A, and CYP17 mRNA expression were higher in thecal cells from PCOS follicles than in size-matched control follicles. In granulosa cells, the mean levels of LH receptor and CYP11A, but not StAR, mRNA expression were higher in PCOS than in control follicles. These data demonstrate that regulatory protein and steroidogenic enzyme mRNAs are overexpressed in thecal and granulosa cells from polycystic ovaries and support the conclusions that the thecal cells are hyperstimulated and the granulosa cells may be prematurely luteinizing.

Adult↗

[Chromatographic separation of ribonucleic acids from bacterial and animal cells on MAB columns].

Practicable and efficient method of the chromatographic separation of ribonucleic acids from animal and bacterial cells on columns with MAB-methylated albumin adsorbed on bentonite basis is developed. Distinct separation of transport and ribosomal RNAs was obtained on MAB columns. Ribosomal RNA from bacterial cells was, in its turn, separated in two fractions. Effect of pH, elution rate and temperature on the efficiency of the fractionation is studied. The method proposed is shown to have a number of advantages as compared with well-known method of RNA fractionation on MAK columns. The essence of the method described is irreversible inactivation of RNAse by AMB, which is confirmed in the series of special experiments.

Animals↗

On the conservation of aminoacyl-transfer ribonucleic acid synthetase genes in Bacillus.

None of the heterologous deoxyribonucleic acid from various bacilli was capable of transforming lysyl- and tryptophanyl-transfer ribonucleic acid (tRNA) synthetase mutants of Bacillus subtilis to wild type. It was concluded that there is little conservation of the aminoacyl-tRNA synthetases even though the tRNA cistrons are conserved genetic functions.

Alleles↗

Covalent attachment of ribonucleic acids to proteins.

As a prerequisite for the synthesis of affinity labels, we describe methods to couple histones to ribonucleic acids. For the synthesis of these covalent hybrid molecules, we used a population of histones H1, H2A, H2B, H3, and H4 from calf thymus and polyadenylic acid with an average chain length of up to 260-280 bases, representing the size of poly(A)-tails from mature mRNAs. Three methods were investigated. (a) Poly(A) containing an 8-N3-A residue was cross-linked to histones by ultraviolet irradiation. (b) The 3'-end of the polynucleotide was connected to a mononucleotide containing an aliphatic amino group, and the resulting poly(A)-derivative was coupled to histones via derivation with a bromoacetyl group. (c) The 3'-end of the polynucleotide was oxidized with sodium periodate and bound covalently to an amino group of the polypeptide. To demonstrate the RNA content of the hybrid molecule, the poly(A) was removed with RNase T2.

Animals↗

Application of a nuclease from rye nucleus for structural studies of plant ribonucleic acids.

A new nuclease (Rn) isolated from rye nucleus was applied for the structural studies of methionine initiator transfer ribonucleic acid and ribosomal 5S rRNA from yellow lupin seeds. The enzyme shows high specificity for some regions of both RNAs. The dihydrouridine and ribothymidine loops which are supposed to be involved in the tertiary interactions of the methionine initiator tRNA were hydrolysed. The anticodon loop is not digested at all. 5S rRNA was digested in single stranded regions (loops). The cleavage pattern of the tRNA and 5S rRNA obtained with Rn enzyme, suggests not only the high specificity toward single stranded regions, but also some dependence on their tertiary structure.

Base Sequence↗