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At least 811 records · Page 45Linked to original sources
PubMed2003

Formation of stable triplexes between purine RNA and pyrimidine oligodeoxyxylonucleotides.

Hybridization properties of oligodeoxyxylonucleotides (OXNs) built from pyrimidine monomers with an inverted 3'-OH group of the furanose have been studied using the gel mobility shift, UV melting and circular dichroism (CD) spectroscopy methods. Pyrimidine OXNs form triple helices with complementary purine RNA in which one OXN is parallel and another is antiparallel with respect to the RNA target. Surprisingly, no duplex formation between the pyrimidine OXNs and purine RNAs is detected. The modified triplexes are stable at pH 7. Their thermal stability depends on the number of C(G-C) triplets and, for G-rich RNA sequences, it is comparable with the stability of native DNA-RNA duplexes. The CD spectra of triplexes formed by OXNs with purine RNA targets are similar to spectra of A-type helices. A pyrimidine OXN having a clamp structure efficiently inhibits reverse transcription of murine pim-1 mRNA in vitro mediated by the Mo-MuLV reverse transcriptase.

Animals↗
PubMed1997

Naegleria nucleolar introns contain two group I ribozymes with different functions in RNA splicing and processing.

We have characterized the structural organization and catalytic properties of the large nucleolar group I introns (NaSSU1) of the different Naegleria species N. jamiesoni, N. andersoni, N. italica, and N. gruberi. NaSSU1 consists of three distinct RNA domains: an open reading frame encoding a homing-type endonuclease, and a small group I ribozyme (NaGIR1) inserted into the P6 loop of a second group I ribozyme (NaGIR2). The two ribozymes have different functions in RNA splicing and processing. NaGIR1 is an unusual self-cleaving group I ribozyme responsible for intron processing at two internal sites (IPS1 and IPS2), both close to the 5' end of the open reading frame. This processing is hypothesized to lead to formation of a messenger RNA for the endonuclease. Structurally, NaGIR2 is a typical group IC1 ribozyme, catalyzing intron excision and exon ligation reactions. NaGIR2 is responsible for circularization of the excised intron, a reaction that generates full-length RNA circles of wild-type intron. Although it is only distantly related in primary sequence, NaSSU1 RNA has a predicted organization and function very similar to that of the mobile group I intron DiSSU1 of Didymium, the only other group I intron known to encode two ribozymes. We propose that these twin-ribozyme introns define a distinct category of group I introns with a conserved structural organization and function.

Animals↗
PubMed1990

In hepatocytes infected with duck hepatitis B virus, the template for viral RNA synthesis is amplified by an intracellular pathway.

During the productive phase of chronic hepadnaviral infections, virion DNA synthesis occurs in the cytoplasm of the infected hepatocyte, but viral RNA is synthesized in the nucleus, apparently from a covalently closed, circular (CCC) viral DNA. J. Tuttleman, C. Pourcel, and J. Summers (1986a, Cell 47, 451-460) have shown that the intracellular levels of CCC DNA can increase during initiation of infection of duck hepatocytes in vitro with duck hepatitis B virus and during long term culture of infected duck hepatocytes in vitro. This amplification of CCC DNA occurs through the reverse transcription pathway. To distinguish between an entirely intracellular process of amplification and amplification due to multiple infections by extracellular virus in the virus producing cultures, suramin was added to the infected cultures to block superinfection. We found that CCC DNA amplification occurred at least as efficiently in the presence of suramin as in its absence. First, there was a net increase in the total amount of CCC DNA in the cultures both in the presence and in the absence of suramin. Second, synthesis of CCC DNA in the presence and absence of suramin was observed by density labeling of this viral DNA by growth of the cultures in medium containing BUdR. Amplification was also demonstrable in the presence of neutralizing duck antibodies. These results support the hypothesis of Tuttleman et al. (1986a) that CCC DNA amplification in chronically infected cultures and, by inference, the mechanism of persistent infection involves primarily intracellular regulatory mechanisms.

Animals↗
PubMedDate not supplied

Role of reverse transcription in the generation of extrachromosomal copia mobile genetic elements.

The Drosophila genetic element copia is one of the best studied eukaryotic transposable sequences. Copia shares structural features with a wide variety of mobile elements in Drosophila, Lepidoptera, yeast and vertebrates, the last class being the retrovirus proviruses. Furthermore, retrovirus-like particles containing copia RNA have been isolated from Drosophila cells and extrachromosomal circular copias with structures closely resembling circular retrovirus proviruses have been isolated and cloned. Therefore, copia-like elements and retroviruses may be members of a class of mobile genetic elements existing throughout the eukaryotic kingdom. Consequently, there has been speculation that retroviruses evolved from transposable elements and, conversely, that copia-like elements transpose as retroviruses or retrovirus-like particles. To date, however, there has been no demonstration that copia RNA is reverse transcribed into copia DNA. The present report describes the isolation of linear extrachromosomal copias whose structure closely resembles the analogous retrovirus provirus linears and whose synthesis is unaffected by inhibitors of the cellular DNA polymerase responsible for chromosomal DNA replication.

Animals↗
PubMed1991

Characterization of elongating T7 and SP6 RNA polymerases and their response to a roadblock generated by a site-specific DNA binding protein.

As a means of generating homogeneous populations of elongation complexes with the RNA polymerases encoded by phages T7 and SP6, transcription has been carried out in vitro on templates associated with the Gln-111 mutant of EcoRI endonuclease. The Gln-111 protein, as a result of a single amino acid substitution at position 111, lacks cleavage function yet shows higher than wild-type affinity for the EcoRI recognition sequence GAATTC. On a series of linear and circular templates associated with Gln-111 protein, blockage of the phage RNA polymerase elongation complex is observed. The 3' endpoint of the major blocked-length RNA species, just 3 bp upstream from the GAATTC, reveals an extremely close approach of polymerase's leading edge to essential contacts between Gln-111 protein and its binding site. In contrast to E. coli RNA polymerase, which is blocked stably and quantitatively by Gln-111 protein (Pavco, P.A. and Steege, D. A. (1990) J. Biol. Chem. 265, 9960-9969), the phage polymerases show substantial levels of readthrough transcription beyond the protein block.

Base Sequence↗
PubMed1983

Rapid purification of plasmid DNA by a single centrifugation in a two-step cesium chloride-ethidium bromide gradient.

A procedure for rapid, preparative purification of plasmid DNA is described and compared with a conventional equilibrium centrifugation method. A discontinuous, two-step CsCl-ethidium bromide gradient is used, with the starting position of the plasmid-containing extract being at the bottom of the tube. During centrifugation in a fixed angle rotor, covalently closed circular plasmid DNA is separated from contaminating protein, RNA, and chromosomal DNA in 5 hr. Plasmids purified by this method are considerably less contaminated with RNA than when purified by a 48-hr equilibrium run in a homogeneous gradient, as determined by agarose gel electrophoresis and 5'-end-labeling studies. Plasmid DNA purified in two-step gradients can be used directly for restriction endonuclease analysis and DNA sequencing.

Autoradiography↗
PubMed2004

An extracellular endodeoxyribonuclease from Streptomyces aureofaciens.

Several extracellular DNases were detected after cultivation of Streptomyces aureofaciens B96 under submerged conditions. These DNases are nutritionally regulated and high content of amino acid nitrogen in cultivation medium repress their production. By varying cultivation conditions, there remained only two extracellular nuclease activities. The major one, extracellular endodeoxyribonuclease SaD I, was purified to homogeneity by ammonium sulfate precipitation, adsorption on Spheron, chromatography on Superose-12P followed by FPLC on MonoQ and final purification on HiTrapQ. The molecular weight of the purified SaD I determined by SDS-PAGE was 31 kDa. The DNase hydrolyses endonucleolytically both double-stranded and single-stranded circular and linear DNA. It does not cleave RNA and does not exhibit phosphodiesterase nor phosphomonoesterase activity. It requires a divalent cation (Zn2+, Co2+, Mn2+, Mg2+) and its activity optimum is at neutral pH (pH 7.2). The optimal temperature for DNA cleavage was 40 degrees C. Activity was strongly inhibited in the presence of phosphate, Hg2+, chelating agents or iodoacetate, but it was stimulated by addition of dimethyl sulphoxide.

Endodeoxyribonucleases↗
PubMed2002

Genetic manipulation of Giardia lamblia.

Giardia lamblia is a flagellated protozoan that infects several species including humans and is a major agent of waterborne outbreaks of diarrhea. G. lamblia is also important in the study of basic eukaryotic molecular biology and evolution; however, it has been difficult to employ standard genetic methods in the study of Giardia. Over the past 6 years, two transfection systems were developed and used for the genetic manipulation of G. lamblia. Both systems allow transient or stable transfection of Giardia and/or foreign genes. The DNA-based transfection system allows electroporation of circular or linear plasmid DNA into trophozoites. The RNA virus-based transfection system requires electroporation of in vitro transcribed RNA into GLV-infected trophozoites. Because G. lamblia is one of the most rudimentary eukaryotes, its processes of transcription, translation and protein transport, as well as its metabolic and biochemical pathways, are of interest. Study of these areas will continue to be advanced using transfection in combination with cellular and molecular tools. Several groups have combined these technologies with other techniques to study protein transport and the transcriptional and post-transcriptional regulation of Giardia genes, including encystation-specific and variant surface protein genes. In addition, coupling antisense techniques with transfection has permitted functional knockout of Giardia metabolic genes, allowing Giardia metabolic pathways to be studied. In the near future, both transfection systems will be potent tools in our investigations of the perplexing questions in Giardia biology.

Animals↗