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PubMedDate not supplied

[Optical measurements of nucleic acids in the presence of phenol and protein impurities].

Spectrophotometric method of determining the presence of phenol admixture in nucleic acid samples is presented. The method is based on determining the optical density ratio at 270 and 240 nm. This ratio is highly sensitive to the presence of phenol in solution. The method of determining nucleic acid concentration in the presence of phenol is also described. It is based on recording circular dichroism values at 265 nm for RNA and 275 nm for DNA. It is shown that the ratio of optical densities of nucleic acid samples at 260 and 280 nm is not a very sensitive criterion of the presence of proteins in solution. A much more sensitive characteristics is the ratio of circular dichroism values at 222 and 265 nm.

DNA↗
PubMed1998

Unintegrated circular HIV-1 DNA in the peripheral mononuclear cells of HIV-1-infected subjects: association with high levels of plasma HIV-1 RNA, rapid decline in CD4 count, and clinical progression to AIDS.

We observed 36 HIV-infected patients to evaluate whether the presence of tandem 2-long terminal repeat circular unintegrated HIV-1 DNA (2-LTR) in peripheral blood mononuclear cells (PBMC) at baseline was associated with acceleration of HIV disease. Detection of 2-LTR at baseline correlated with high plasma HIV-1 RNA levels (p < .01), recovery of culturable HIV-1 from plasma (p = .02), and progression to AIDS during follow-up (p = .01). More patients with 2-LTR (68%) than without 2-LTR (31%) had a decline in CD4 levels of >50 cells/mm3 over the first 18 months of follow-up (p = .04), and the average annual CD4 decline was 35% in patients with 2-LTR compared with 16% in those without 2-LTR (p = 0.06). Detection of 2-LTR in PBMC at baseline was an independent predictor of high plasma HIV-1 RNA levels and subsequent CD4 cell decline in this cohort of patients with predominantly nonsyncytium-inducing (NSI) isolates at baseline. The presence of 2-LTR in PBMC appears to be reflective of ongoing HIV-1 replication, as measured by plasma HIV-1 RNA levels, and identifies persons at risk for immunologic and clinical decline.

Adult↗
PubMedDate not supplied

Incorporation of two anthraquinonylmethyl groups into the 2'-O-positions of oligonucleotides: increased affinity and sequence specificity of anthraquinone-modified oligonucleotides in hybrid formation with DNA and RNA.

Oligonucleotide 15-mers containing one or two anthraquinonylmethyl groups at specified sugar residues have been prepared on an automated DNA/RNA synthesizer by using 5'-O-dimethoxytrityl 2'-O-(2-anthraquinonylmethyl)uridine 3'-O-(2-cyanoethyl)-N, N-diisopropylphosphoramidite. The purification of the modified oligonucleotides was done with denaturing polyacrylamide gel electrophoresis. The base compositions and the presence of anthraquinone group(s) in the oligonucleotides were verified with enzymatic digestion (snake venom phosphodiesterase and alkaline phosphatase) analysis and UV-vis spectral measurements. The UV melting behaviors indicate that all the oligonucleotides with anthraquinone group(s) can bind to both their complementary DNA and RNA in a manner similar to that of the unmodified oligonucleotide. All the oligonucleotides possessing anthraquinone group(s) have higher affinity for both DNA and RNA segments when compared with the unmodified oligonucleotide. The oligomer containing two anthraquinone substituents at sites separated by four nucleotides instead of six exhibits the highest affinity for both the complementary DNA and RNA. The stabilizing effect can be translated into a free energy cost of 7.1 kcal/mol for the DNA hybrid and 3.6 kcal/mol for RNA. It has been shown through mismatch/Tm studies that modification of the oligonucleotide by anthraquinone groups does not alter the sequence specificity in binding to a RNA segment.

Anthraquinones↗
PubMed1990

Elongation by Escherichia coli RNA polymerase is blocked in vitro by a site-specific DNA binding protein.

As a means of determining how elongating RNA polymerase responds to a protein in its path, transcription has been carried out in vitro with the purified Escherichia coli enzyme on templates associated with a sequence-specific DNA binding protein. The major RNA species generated is the length expected from RNA polymerase which has transcribed to the position of the bound protein and is unable to elongate further. The binding proteins used are two mutants of the EcoRI endonuclease which are defective in cleavage function but retain high affinity for the wild-type recognition sequence (Wright, D. J., King, K., and Modrich, P. (1989) J. Biol. Chem. 264, 11816-11821). Blockage of RNA polymerase occurs on linear and circular templates and, although efficient with both proteins, is more effective for the EcoRI derivative with the slower dissociation rate. The protein-blocked transcription complexes are stable over time and remain in an active form, resuming elongation when the blocking protein is displaced by an increase in ionic strength. These paused ternary complexes, if treated with the termination factor rho, undergo release. The 3' ends of the blocked-length RNAs from DNAs of distinct sequences reveal that the ternary complexes are positioned at a constant distance from the protein block, 14 nucleotides upstream of the EcoRI recognition sequence. This information is combined with exonuclease III footprinting data to position the 3' end of the nascent RNA chain in the ternary complex quite near (approximately 7 nucleotides) the leading edge of RNA polymerase.

Bacteriophage lambda↗
PubMed1975

DNA-dependent RNA polymerase from Pseudomonas BAL-31. II. Transcription of the allomorphic forms of bacteriophage PM2 DNA.

Transcription of the supercoiled form (I) and the relaxed circular form (II) of bacteriophage PM2 DNA was studied utilizing the DNA-dependent RNA polymerase from its host, Pseudomonas BAL-31. Transcription of both templates is continuous for up to 2 hr, but proceeds at a two-fold higher rate on I than on II. This difference is mainly due to a 2.2-fold higher rate of chain initiation on I. When rifampicin (Rif) is added ater 10 min of synthesis, (1) transcription of II ceases by 30 min with a maximum product length of 7000 nucleotides (number average) being produced; (2) transcription of I continues with little rate reduction and with the product reaching 16,000 nucleotides (number average) by 2 hr. Sucrose gradient analysis shows that the product of II achieves maximum size 20 min after Rif addition and sediments in three peaks of 24, 33, and 39 S (approximately one-third, two-thirds, and one genome lengths). The product of I has a heterogeneous distribution and grows continuously with a large fraction reacting greater than 3 genome lengths by 90 min. The same differences in synthesis kinetics, Rif inhibition, and product size distribution are observed when I and II are transcribed by Escherichia coli RNA polymerase. These experiments show that (i) PM2 form I DNA is transcribed mainly by a process of continuous chain elongation, with little chain termination occurring; (ii) PM2 form II is transcribed by a process of continuous chain initiation, elongation, and termination of yield discrete products. Thus, the tertiary structure of circular DNA influences chain termination by RNA polymerase.

Bacteriophages↗
PubMed2005

Structure of a folding intermediate reveals the interplay between core and peripheral elements in RNA folding.

Though the molecular architecture of many native RNA structures has been characterized, the structures of folding intermediates are poorly defined. Here, we present a nucleotide-level model of a highly structured equilibrium folding intermediate of the specificity domain of the Bacillus subtilis RNase P RNA, obtained using chemical and nuclease mapping, circular dichroism spectroscopy, small-angle X-ray scattering and molecular modeling. The crystal structure indicates that the 154 nucleotide specificity domain is composed of several secondary and tertiary structural modules. The structure of the intermediate contains modules composed of secondary structures and short-range tertiary interactions, implying a sequential order of tertiary structure formation during folding. The intermediate lacks the native core and several long-range interactions among peripheral regions, such as a GAAA tetraloop and its receptor. Folding to the native structure requires the local rearrangement of a T-loop in the core in concert with the formation of the GAAA tetraloop-receptor interaction. The interplay of core and peripheral structure formation rationalizes the high degree of cooperativity observed in the folding transition leading to the native structure.

Bacillus subtilis↗
PubMed1985

Oligomerization of intervening sequence RNA molecules in the absence of proteins.

The intervening sequence RNA excised from the ribosomal RNA precursor of Tetrahymena forms linear and circular oligomers when exposed to a heating-cooling treatment in vitro. The reactions require no protein or external energy source. Oligomerization is different from other self-catalyzed reactions of the intervening sequence RNA in that it involves intermolecular rather than intramolecular recombination, producing RNA molecules that are substantially larger than the original. The observation that RNA molecules can catalyze their own oligomerization has possible implications for the evolution of chromosomes and for the replicative cycle of plant viroids and virus-associated RNA's.

Animals↗
PubMedDate not supplied

[Enhanced transcription of genes of the intracisternal A particles and B2 elements in mouse tumors].

A comparison of the expression of two mobile genetic elements A1 and B2 was studied in normal and tumor tissues. The A1 element is a chromosomal homolog of IAP genes, and B2 is a short ubiquitous repetitive sequences of the mouse genome. These sequences were earlier cloned in our laboratory and in this study were used as probes in hybridization experiments with RNA isolated from different mouse tumor and normal tissues. Both elements were efficiently transcribed in tumor cells. The level of expression of A1 sequences in tumors was 100-200 times higher than in normal tissues. The amount of B2 small cytoplasmic RNA significantly varied in different normal tissues. The content of this RNA was much higher in tumors. Closed circular DNA molecules containing IAP sequences were found in Ehrlich carcinoma cells. These DNA molecules are considered as intermediate forms of the mobile elements. The role of these mobile elements in the regulation of RNA expression and tumor progression is discussed.

Animals↗
PubMed1997

Synthesis and two-dimensional electrophoretic analysis of mixed populations of circular and linear RNAs.

Spontaneous cleavage of the less abundant form of tobacco ringspot virus satellite RNA is readily reversible. Capitalizing on earlier observations by Feldstein and Bruening that small 'mini-monomer' RNAs derived from this molecule and containing little more than covalently attached ribozyme and substrate cleavage products are able to efficiently circularize, we have constructed a series of self-circularizing RNAs of precisely known size. Mixtures of linear and circular RNAs synthesized in vitro and containing 225-1132 nt could be completely resolved using a novel two-dimensional denaturing polyacrylamide gel electrophoresis system. Similar analyses of a complex mixture of coconut cadang-cadang viroid RNAs revealed the presence of relatively large amounts of a previously undescribed 'fast-slow' heterodimeric RNA species in infected palms. Only a single DNA template is required to prepare each pair of circular and linear RNA markers.

Base Sequence↗
PubMedDate not supplied

Circular rDNA replicons persist in Tetrahymena thermophila transformants synthesizing GGGGTC telomeric repeats.

Site-directed mutagenesis of the telomerase RNA from Tetrahymena thermophila was used previously to demonstrate the templating function of a sequence within this RNA; this sequence specifies the sequence of telomeric DNA in vivo. The possible functional importance of a phylogenetically conserved nucleotide outside the telomerase RNA template region was investigated by a similar experimental approach. The telomerase RNA gene was altered by site-directed mutagenesis, cloned in a circular selectable transformation vector consisting of an rRNA gene carrying a selectable drug resistance marker, and introduced into macronuclei of vegetatively dividing Tetrahymena thermophila by microinjection. Changing an invariant A to U at position 16 of the telomerase RNA (A16U) had no effect detectable by phenotype on telomerase function in vivo. However these experiments showed that a telomerase template alteration that dictates the synthesis of the mutant telomeric DNA sequence GGGGTC leads to a profound change in the population of rDNA replicons. The addition of GGGGTC mutant repeats leads to selective pressure for the loss of high copy linear rDNA, and the rRNA genes are maintained in the form of the circular rDNA replicons introduced during transformation.

Animals↗
PubMed2025

Clinically relevant pseudoexons of the GALNS gene and their antisense-based correction.

BACKGROUND: Biallelic pathogenic variants in the GALNS gene lead to Mucopolysaccharidosis Type IVA (MPS IVA), a rare lysosomal storage disorder. GALNS encodes the enzyme N-acetylgalactosamine-6-sulfatase, whose deficiency causes accumulation of glycosaminoglycans and leads to a broad spectrum of clinical manifestations primarily affecting the osteoarticular system. Several studies have shown that, in 10%-15% of patients with the biochemical phenotype of MPS IVA, standard molecular genetic testing fails to identify one or both causative variants in the GALNS gene. METHODS: We performed an in-depth investigation of GALNS' splicing, with a special focus on deep-intronic mutations that lead to activation of pseudoexons (PEs). Using bioinformatic tools, we analyzed all deep-intronic variants in GALNS available in public databases and subjected the most relevant ones to in vitro analyses using minigenes. RESULTS: We characterized eight PE-activating variants, one of which (c.121-210C&#x2009;>&#x2009;T) represents a recurrent pathogenic variant which has long been hidden behind the mask of a polymorphic variant. In addition, we demonstrate that GALNS' splicing can produce a diverse range of mRNA isoforms containing so-called wild-type PEs, which are present at low levels as part of non-productive splicing, and weak canonical exons which are prone to skipping. We show that PE-activating variants cluster within wild-type PEs, highlighting the need for closer scrutiny of these regions during genetic testing. Finally, we applied modified U7 small nuclear RNAs and circular RNAs to efficiently block the identified PEs and pave the way for personalized antisense-based therapy for MPS IVA patients. CONCLUSION: The results of this study expand the understanding of GALNS gene splicing, indicating hotspots for splicing mutations. The presented data not only help to increase the diagnostic yield for MPS IVA but also unveil new therapeutic approaches for a number of MPS IVA patients.

Humans↗
PubMed1991

Rapid isolation of plasmid DNA by LiCl-ethidium bromide treatment and gel filtration.

We established a simple and rapid plasmid DNA purification method. Crude plasmid DNA preparations are treated with 4 M LiCl in the presence of 0.6 mg/ml ethidium bromide to precipitate RNA and proteins contained in the DNA preparations. After removal of RNA and protein precipitates, the supernatant is filtered through a Sepharose CL6B column to remove low-molecular-weight contaminants. This procedure takes only 30 min and provides pure plasmid DNA preparations that consist mainly of covalently closed circular plasmid DNA but have no detectable RNA and protein. The purified DNA preparations are susceptible to various six- and four-base-recognition restriction endonucleases, T4 DNA ligase, the Klenow fragment of DNA polymerase I, and T7 and Taq DNA polymerase. Since no special equipment is needed for this purification method, 20 or more samples of microgram to milligram levels can be treated in parallel.

Chemical Precipitation↗
PubMed1984

Effect of template conversion from the B to the Z conformation on RNA polymerase activity.

Transition from the right-handed B to the left-handed Z conformation of DNA was studied by circular dichroism in parallel with the ability of the DNA to support RNA synthesis with Escherichia coli RNA polymerase. Since the B to Z transition is generally induced by a chemical agent, a definitive demonstration that a change in activity is due to the conformational change, and not to the agent itself or to other factors, requires the clear-cut correlation of template activity and conformation under a variety of conditions that result in conformational change. Such correlation was achieved by following the [Co(NH3)6]3+-induced transition of poly(dG-dC) X poly(dG-dC) and poly(dG-dm5C) X poly(dG-dm5C) and the Mg2+-induced transition of poly(dG-dm5C) X poly(dG-dm5C). In addition, conditions were chosen to minimize possible aggregation. In each of these three systems, the B to Z conformational transition was accompanied by a substantial decrease in transcription activity. While the conversion from B to Z of poly(dG-dm5C) X poly(dG-dm5C) is induced by a 25-fold lower concentration of [Co(NH3)6]3+ than that required for the conversion of unmethylated polymer, in both cases the RNA polymerase activity is decreased at the same cation concentration as that producing the conformational transition. Neither [Co(NH3)6]3+ nor Mg2+ inhibits RNA synthesis with control templates that are not converted to Z under the same conditions, such as poly(dA-dT) X poly(dA-dT) or calf thymus DNA with [Co(NH3)6]3+ or poly(dG-dC) X poly(dG-dC) with Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗
PubMed2002

Alpha-LNA (locked nucleic acid with alpha-D-configuration): synthesis and selective parallel recognition of RNA.

Alpha-LNA is presented as a stereoisomer of LNA (locked nucleic acid) with alpha-D-configuration. Three different approaches towards the thymine alpha-LNA monomer as well as the 5-methylcytosine alpha-LNA monomer are presented. Different alpha-LNA sequences have been synthesised and their hybridisation with complementary DNA and RNA has been evaluated by means of thermal stability experiments and circular dichroism spectroscopy. In a mixed pyrimidine sequence, alpha-LNA displays unprecedented parallel-stranded and selective RNA binding. Furthermore, a remarkable selectivity for hybridisation with RNA over DNA is indicated.

Circular Dichroism↗