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At least 1,531 records · Page 85Linked to original sources
PubMed2000

Extraction of total RNA from a high pigment content plant. Marigold (Tagetes erecta).

The Mexican marigold (Tagetes erecta) produces inflorescences of intense yellow color that contain high levels of xanthophylls, particularly lutein, which makes it a suitable model for the study of carotenoid biosynthesis and regulation throughout the development of the inflorescences. However, these studies require the recovery of total RNA from floral buds and inflorescences at different developmental stages, each of which presents specific extraction problems. Four protocols were tested, but only through the modification of one of them was it possible to obtain total RNA of sufficient quality and quantity to perform RT-PCR and Northern blots and to construct a cDNA library. This article presents the modified protocol for the recovery of total RNA from carotenoid-rich plant tissues.

Asteraceae↗
PubMed2003

Alphavirus cDNA-based expression vectors: effects of RNA transcription and nuclear export.

The construction of layered DNA-RNA replicons has facilitated and expanded the use of alphavirus vectors to vaccine development, construction of packaging cell lines and long-term heterologous gene expression. In these vector systems, the alphavirus replicon is under the control of a strong RNA polymerase II promoter and replicon RNA is transcribed from DNA before transport to the cytoplasm. Efficient RNA amplification catalyzed by the viral replicase results in high levels of mRNA and the recombinant protein. Recently, we developed a temperature-regulated Sindbis replicon-based DNA expression system characterized by a linear increase of expression upon decrease of the temperature from 37 degrees C to 29 degrees C. Modifications known to affect transcription and nuclear export of RNA led to a 5-fold increase in expression in BHK cells and up to over 80-fold increase in CHO cells and BF fibroblasts in transient transfection experiments. Furthermore, reducing cell proliferation resulted in a further 2- to 3-fold higher expression. While increased expression per cell was responsible for some of the enhanced production, it was primarily the number of expressing cells that made the difference in most cell lines. Further experiments indicated that a threshold amount of replicon RNA had to reach the cytoplasm in order for replication to occur. Thus, alterations that improve transcription, nuclear export and stability of the RNA had a significant impact on protein production in the pCytTS expression system and probably in other layered DNA-based viral vectors. Furthermore the results indicate that RNA replication is differentially regulated in DNA layered RNA replicons versus viral infection.

Active Transport, Cell Nucleus↗
PubMed1997

Posttranscriptional modification of tRNA in psychrophilic bacteria.

Posttranscriptional modification in tRNA is known to play a multiplicity of functional roles, including maintenance of tertiary structure and cellular adaptation to environmental factors such as temperature. Nucleoside modification has been studied in unfractionated tRNA from three psychrophilic bacteria (ANT-300 and Vibrio sp. strains 5710 and 29-6) and one psychrotrophic bacterium (Lactobacillus bavaricus). Based on analysis of total enzymatic hydrolysates by liquid chromatography-mass spectrometry, unprecedented low amounts of modification were found in the psychrophiles, particularly from the standpoint of structural diversity of modifications observed. Thirteen to 15 different forms of posttranscriptional modification were found in the psychrophiles, and 10 were found in L. bavaricus, compared with approximately 29 known to occur in bacterial mesophiles and 24 to 31 known to occur in the archaeal hyperthermophiles. The four most abundant modified nucleosides in tRNA from each organism were dihydrouridine, pseudouridine, 7-methylguanosine, and 5-methyluridine. The molar abundances of the latter three nucleosides were comparable to those found in tRNA from Escherichia coli. By contrast, the high levels of dihydrouridine observed in all three psychrophiles are unprecedented for any organism in any of the three phylogenetic domains. tRNA from these organisms contains 40 to 70% more dihydrouridine, on average, than that of the mesophile E. coli or the psychrotroph L. bavaricus. This finding supports the concept that a functional role for dihydrouridine is in maintenance of conformational flexibility of RNA, especially important to organisms growing under conditions where the dynamics of thermal motion are severely compromised. This is in contrast to the role of modifications contained in RNA from thermophiles, which is to reduce regional RNA flexibility and provide structural stability to RNA for adaptation to high temperature.

Base Composition↗
PubMed1994

The apolipoprotein B messenger RNA editing enzyme.

The editing of apolipoprotein (apo)B messenger RNA (mRNA) involves a novel C to U modification, which creates an in-frame stop-translation codon, thereby generating the carboxyl-terminal of apoB48. The 27 kDa catalytic subunit of the editing enzyme has been cloned and established to be a zinc-containing cytidine deaminase. The catalytic subunit is guided to the editing site by a second targeting subunit or subunits. A candidate for the targeting subunit is a 60 kDa protein that can be UV crosslinked to the sequence UGAU, which is part of a motif downstream of the editing site that is essential for editing.

Amino Acid Sequence↗
PubMed1978

Synthesis of modified nucleoside 3',5'-bisphosphates and their incorporation into oligoribonucleotides with T4 RNA ligase.

A simple procedure is described to prepare nucleoside 3'(2'),5'-bisphosphates from the corresponding nucleosides with the use of pyrophosphoryl chloride. This method is rapid, gives nearly quantitative yields and, most importantly, can be used for a variety of nucleosides with base and sugar modifications. Since 3',5'-bisphosphates are donors in the T4 RNA ligase reaction, a single residue can be enzymatically attached to the 3' end of oligoribonucleotides. By these procedures, five different ring-modified nucleosides and one sugar-modified nucleoside were incorporated onto the 3' end of (Ap)3C. In two cases, an additional step of synthesis with RNA ligase resulted in the modified nucleotide being located in an internal position in the oligonucleotide. Thus, a general method for the synthesis of oligoribonucleotides containing modified nucleosides is outlined. Since many of the modified nucleosides are fluorescent, oligomers containing them should be useful in a variety of physical and biochemical studies.

Coliphages↗
PubMed1981

Quantitative EEG and neurochemical aspects of memory and learning.

Different, established or putative animal models of learning were studied by electrophysiological (EEG) and neurochemical methodologies. The training of rats to new behavioral patterns proved to result in the stimulation of total RNA synthesis rate in specific brain structures, as well as in modifications of the EEG organization of hippocampus. This two-fold approach to the assessment of modifications in learning-involved brain structures was extended to the study of mirror focus and kindling, to verify the suitability of these phenomena as experimental models of learning. Reduced (3H) uridine incorporation and proportion of poly (A)-associated RNA were found in mirror focus in comparison with control regions. These variations seem related to brain damage rather than be congruent with learning-related modifications. Further studies are necessary to verify whether the kindling phenomena are to be equated to learning models.

Amygdala↗
PubMed1982

Translation of mRNA from phytohemagglutinin-stimulated human lymphocytes: characterization of interferon mRNAs.

Human lymphocytes obtained by cytapheresis were stimulated in spinner culture conditions by nonpurified PHA in order to study the production of gamma interferon, and the characterization of IFN-gamma mRNA. Titers of interferon prepared in 0.6 to 4 1 batches, varied in 20 preparations from 8,000 to 32,000 units/ml. This interferon was unstable at pH 2: the residual antiviral activity after 20 h treatment was less than 3%. Antibodies raised against gamma interferon from Con A and SEA-stimulated lymphocytes neutralized the interferon induced by PHA, indicating that all three preparations are antigenically related. Poly(A)RNA from control, noncultivated lymphocytes and from lymphocytes stimulated by PHA for 18 h were translated in reticulocyte lysates and analysed by polyacrylamide gel electrophoresis. The pattern of synthesized polypeptides was different suggesting modifications in the population of mRNA. When total poly(A)RNA was inoculated into Xenopus Laevis oocytes, interferon activity was found with both, control and stimulated mRNAs although only at low levels in the control. After sucrose gradient fractionation of poly(A)RNA, each fraction was inoculated into oocytes and interferon activity measured in the oocyte bathing medium. A low level was synthesized by the RNA fractions around 28 S from control as well as from stimulated lymphocytes. These interferons were not neutralized by anti-IFN-alpha or anti-IFN-gamma sera but they were neutralized by anti-IFN-beta serum. Only the 16 S RNA fraction from PHA-stimulated lymphocytes induced high levels of interferon in oocytes. This interferon has been characterized as gamma interferon. Each fraction obtained from sucrose gradients on poly(A)RNA from control and PHA-stimulated lymphocytes was translated in reticulocyte lysate. Gel analysis of the products showed striking differences when the same fraction of both RNAs were compared. Concerning particularly the 16 S RNA from PHA-stimulated lymphocytes, where gamma interferon mRNA was present, polypeptides ranged from 15 to 55 K with a bulk around 45 K, indicating heterogeneous RNA molecules.

Animals↗
PubMed2005

Essential amino acids of the hantaan virus N protein in its interaction with RNA.

The nucleocapsid (N) protein of hantavirus encapsidates viral genomic and antigenomic RNAs. Previously, deletion mapping identified a central, conserved region (amino acids 175 to 217) within the Hantaan virus (HTNV) N protein that interacts with a high affinity with these viral RNAs (vRNAs). To further define the boundaries of the RNA binding domain (RBD), several peptides were synthesized and examined for the ability to bind full-length S-segment vRNA. Peptide 195-217 retained 94% of the vRNA bound by the HTNV N protein, while peptides 175-186 and 205-217 bound only 1% of the vRNA. To further explore which residues were essential for binding vRNA, we performed a comprehensive mutational analysis of the amino acids in the RBD. Single and double Ala substitutions were constructed for 18 amino acids from amino acids 175 to 217 in the full-length N protein. In addition, Ala substitutions were made for the three R residues in peptide 185-217. An analysis of protein-RNA interactions by electrophoretic mobility shift assays implicated E192, Y206, and S217 as important for binding. Chemical modification experiments showed that lysine residues, but not arginine or cysteine residues, contribute to RNA binding, which agreed with bioinformatic predictions. Overall, these data implicate lysine residues dispersed from amino acids 175 to 429 of the protein and three amino acids located in the RBD as essential for RNA binding.

Amino Acid Sequence↗
PubMed1994

Enhanced competitiveness of tomato bushy stunt virus defective interfering RNAs by segment duplication or nucleotide insertion.

We have analyzed atypical tomato bushy stunt virus defective interfering (DI) RNA species which accumulated during a passage series in protoplasts. We present a rationale for the order of appearance of these molecules and show, using competition assays, that either segment duplication or single nucleotide insertion can enhance DI RNA competitiveness. Possible mechanisms for the introduction of the modifications observed in these DI RNAs are discussed.

Base Sequence↗
PubMed2001

Chronic ethanol consumption and liver glycogen synthesis.

Chronic ethanol consumption results in a dramatic decrease in liver glycogen concentrations, which could be related to either a depressed rate of synthesis or an increased rate of breakdown. Earlier studies suggested that there is not an increase in the rate of glycogenolysis as glycogen phosphorylase activities are not elevated. In the present study it was observed that the incorporation of radiolabeled glucose into glycogen was significantly depressed in hepatocytes from ethanol-fed rats under both anaerobic and aerobic conditions. Chronic ethanol consumption decreased the total glycogen synthase (a + b) activity, which correlated closely with a loss in glycogen synthase protein. However, glycogen synthase messenger RNA levels were not depressed, which indicated posttranscriptional modifications affecting both activity and protein levels. The concentration of glucose transporter 1 was also decreased due to ethanol consumption, but glucose transporter 2 levels were not altered. This latter result suggests that glucose transport in the perivenous region of the liver lobule may be decreased in chronic ethanol consumers. The alterations in glucose transport protein and glycogen synthesis observed in this study may contribute to lowered glycogen synthesis, but do not appear to account for the magnitude of the decreases in glycogen levels and rate of synthesis. Indeed, ethanol effects on glycogen metabolism are likely to be exerted at several levels, including posttranslational modulation of enzyme activities.

Alcoholism↗
PubMed1993

Homology with Saccharomyces cerevisiae RNA14 suggests that phenotypic suppression in Drosophila melanogaster by suppressor of forked occurs at the level of RNA stability.

The suppressor of forked [su(f)] locus of Drosophila melanogaster encodes at least one cell-autonomous vital function. Mutations at su(f) can affect the expression of unlinked genes where retroviral-like transposable elements are inserted. Changes in phenotype are correlated with changes in mRNA profiles, indicating that su(f) affects the production and/or stability of mRNAs. We have cloned the su(f) gene by P-element transposon tagging. Alterations in the DNA map of eight lethal alleles were detected in a 4.3-kb region. P-element-mediated transformation using a fragment including this interval rescued all aspects of the su(f) mutant phenotype. The gene is transcribed to produce a major 2.6-kb RNA and minor RNAs of 1.3 and 2.9 kb, which are present throughout development, being most abundant in embryos, pupae, and adult females. The major predicted gene product is an 84- kD protein that is homologous to RNA14 of Saccharomyces cerevisiae, a vital gene where mutation affects mRNA stability. This suggests that phenotypic modification by su(f) occurs at the level of RNA stability.

Amino Acid Sequence↗
PubMed2004

Path to equality strewn with roX.

Male flies hypertranscribe most genes along their single X chromosome to match the output of females with two X chromosomes. This is mediated by chromatin modifications carried out by the MSL complex composed of noncoding roX RNA and at least five MSL proteins. New results indicate that one of these subunits, the MOF acetyltransferase, not only acts on histone H4, but on itself and MSL3. Cycles of covalent modifications of the MSL subunits may determine the proper level of hypertranscription or control cis spreading along the chromosome. The MSL complex binds to the roX genes, the very source of the RNA component of the complex. New details of how this interaction occurs hint at a possible autoregulatory function. Finally, despite intensive efforts, the molecular mechanism by which the MSL complex distinguishes the X from the autosomes remains a mystery. The MSL complex is able to spread epigenetically from the site of roX transcription, and recent work has defined the conditions that control local cis spreading. However, it is equally clear that soluble MSL complex can distinguish the X chromosome from autosomes. Reconciling all these findings into a unified model presents a challenge.

Animals↗
PubMed1978

Structure and function of 5S ribosomal ribonucleic acid from Torulopsis utilis. IV. Detection of exposed guanine residues by chemical modification with kethoxal.

The reaction of Torulopsis (Candida) utilis 5S ribosomal RNA with kethoxal (beta-ethoxy-alpha-ketobutyraldehyde) was studied in an attempt to identify the exposed guanine residues. At most 7-8 out of 32 guanine residues in T.utilis 5S RNA were kethoxalated after reaction at 37 degrees C for 4 h in the presence of magnesium ions. Localization of the kethoxalated guanine residues in T.utilis 5S RNA was achieved by sequence analyses of RNase T1 digests of the kethoxalated 5S RNA. These analyses showed that residues G37, G57, G91, and some of the three guanine residues G80, G82, and G85, are the most accessible sites. Residues G30, G41, and G49 also reacted with kethoxal though less strongly. These results are for the most part compatible with our secondary structure model for T.utilis 5S 5S RNA (Nishikawa and Takemura (1974) J. Biochem. 76, 935-947). However, partial formation of some hydrogen bonds within the loop region of the model seems to be necessary to explain the inaccessibility of residue G101 to kethoxal. The results are also discussed in comparison with those of similar studies on E.coli 5S RNA.

Aldehydes↗
PubMed1990

Yeast precursor ribosomal RNA. Molecular cloning and probing the higher-order structure of the internal transcribed spacer I by kethoxal and dimethylsulfate modification.

Plasmids were constructed in which a HindIII fragment of rDNA (6.4 x 10(3) base-pairs) was inserted into vectors pGEM-1 and 2 in both orientations. The DNA insert encoded the yeast 35 S precursor rRNA beginning 180 bases upstream from the 5' end of the mature 18 S rRNA and ending 289 bases beyond the 3' end of the mature 25 S rRNA. The precursor rRNA molecules produced in vitro consisted of 6430 nucleotides, with about 15 residues derived from the Gemini vector on both ends. The general extent of secondary structure of the precursor rRNA was examined by ethidium fluorescence and compared to that of the mature rRNAs. Both precursor and mature rRNAs responded similarly to changes in magnesium ion concentration and to digestion by cobra venom and T1 ribonucleases. The higher-order structure of the internal transcribed spacer-1 (ITS-1) region of the 35 S rRNA molecule was further examined by kethoxal and dimethylsulfate modifications and primer extension. Accessible adenine and guanine residues were located by primer extension analysis with avian myeloblastosis virus reverse transcriptase. On the basis of experimental data and computer-generated structures, a secondary structure model was proposed for the ITS-1 region. In this model, six hairpin stems involving adjacent nucleotides are present. A long-range interaction between nucleotides at the middle of the ITS-1 region and an, as yet, unidentified sequence located at another region of the precursor rRNA is suggested also. A candidate for this interacting sequence is that previously proposed, on a theoretical basis, to be involved in the removal of the precursor 18 S rRNA species for 35 S precursor molecule.

Aldehydes↗
PubMed2004

Modified bases in RNA reduce secondary structure and enhance hybridization.

Secondary structure in RNA targets is a significant barrier to short DNA probes. However, when such targets are the end product of an in vitro amplification scheme, it is possible to carry out transcription in the presence of nucleoside triphosphate analogues that reduce secondary structure of the RNA without impairing subsequent hybridization. Here we show that nucleoside triphosphates of 2-aminoadenine (nA) and 2-thiouracil (sU) are taken up by T7 RNA polymerase and that the resulting RNA possesses reduced secondary structure and improved accessibility to DNA probes. The hybridization properties of short RNA transcripts were studied using a new gel mobility shift assay from which melting temperatures were determined. RNA hairpins that contained nA and sU were able to hybridize to DNA probes under conditions where the unmodified hairpins did not. DNA-RNA hybrids that contained nA and sU in the RNA strand exhibited enhanced specificity, increased stability, and greater equality of base pairing strength than the same hybrids without modifications. Substitution of guanine (G) with inosine (I) further reduced secondary structure, but RNA with this base hybridized nonselectively. The high stability of nA-T and A-sU base pairs in DNA-RNA hybrids, combined with the destabilizing effect of the nA-sU couple in RNA targets, accounts for the improved hybridization properties. These results suggest that incorporation of nA and sU during in vitro transcription is a promising strategy for enhancing the performance of oligomeric DNA probes with an RNA target.

2-Aminopurine↗
PubMed2003

RT-PCR of long prokaryotic operon transcripts without DNase treatment.

RT-PCR is a powerful technique used in the amplification and detection of rare mRNAs. However, one of the most serious drawbacks of this method is the amplification of false-positive products due to DNA contamination in the RNA samples. This pitfall is particularly hard to overcome when RNA from prokaryotic origin is used. We present here a modification of the EXACT RT-PCR method that was successfully employed in the amplification of the low abundant full-length polycistronic pst operon mRNA of Escherichia coli. No DNase treatment of the RNA template is required, but unlike the original EXACT RT-PCR, a hybrid primer that is not composed of oligo(dT) was used. A nonhomologous sequence was incorporated at the reverse transcription step into the 5' end of the first-strand cDNA by means of the hybrid primer. For the PCR, a gene-specific primer and a second primer identical to the nonhomologous portion of the hybrid primer were used. To avoid amplification of genomic DNA, the hybrid-primer molecules that were not incorporated into the first-strand cDNA were removed by RNase H treatment followed by ultrafiltration.

ATP-Binding Cassette Transporters↗