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At least 199 records · Page 11Linked to original sources
PubMed2001

Use of a vector based on Potato virus X in a whole plant assay to demonstrate nuclear targeting of Potato spindle tuber viroid.

Potato spindle tuber viroid (PSTVd) is a covalently closed circular RNA molecule of 359 nucleotides that replicates within the nucleus of host cells. To determine how this small, highly structured RNA enters the nucleus, we have developed a virus-based, whole plant in vivo assay that uses green fluorescent protein (GFP) as the reporter molecule. The coding region of GFP was interrupted by insertion of an intron derived from the intervening sequence 2 of the potato ST-LS1 gene. A cDNA copy of the complete PSTVd genome was, in turn, embedded within the intron, and this construct was delivered into Nicotiana benthamiana plants via a vector based on Potato virus X. The intron-containing GFP subgenomic RNA synthesized during virus infection cannot produce a functional GFP unless the RNA is imported into the nucleus, where the intron can be removed and the spliced RNA returned to the cytoplasm. The appearance of green fluorescence in leaf tissues inoculated with constructs containing a full-length PSTVd molecule embedded in the intron indicates that nuclear import and RNA splicing events did occur.

Active Transport, Cell Nucleus↗
PubMed1984

A replication cycle for viroids and other small infectious RNA's.

Experimental data concerning viroid-specific nucleic acids accumulating in tomato plants establish, together with earlier studies, the major features of a replication cycle for viroid RNA in plant cells. Many features of this pathway, which involves multimeric strands of both polarities, may be shared by other small infectious RNA's including certain satellite RNA's and "virusoid" RNA's which replicate in conjunction with conventional plant viruses. The presence, in host plans, of an elaborate machinery for replicating these disease agents suggests a role for endogenous small RNA's in cellular development.

Adenosine Triphosphate↗
PubMed1989

Export and intercellular transfer of DNA via membrane blebs of Neisseria gonorrhoeae.

Naturally elaborated membrane bleb material is frequently observed in cultures of Neisseria gonorrhoeae. This material was purified and analyzed for protein, lipopolysaccharide, and nucleic acid content. The electrophoretic protein profiles of two bleb-rich fractions, called BI and BII, were distinct, with only BII containing lipopolysaccharide and outer membrane proteins I and III. Both fractions contained RNA, circular DNA, and linear DNA. Exogenous pancreatic DNase I appeared to hydrolyze all bleb-associated DNA in fraction BI and the linear DNA in fraction BII. The circular DNA molecules associated with fraction BII resisted digestion. Electron microscopy of the bleb fractions verified their DNA content. Fixing blebs with glutaraldehyde before mounting them for microscopy prevented release of internal DNA. Such fixation produced little change in the micrographs of BI; however, only traces of DNA were observed in fixed BII preparations. Incubation of wild-type gonococci in mixtures of DNase and blebs purified from antibiotic-resistant strains resulted in efficient exchange of penicillinase-specifying R plasmids. Recipients incorporated plasmids independently of endogenous and exogenous chromosomal streptomycin resistance markers. These in vitro results suggest that bleb formation by N. gonorrhoeae may serve to transfer plasmids intercellularly in vivo, perhaps constituting a previously unexplored genetic exchange mechanism in these bacteria.

Bacterial Outer Membrane Proteins↗
PubMed1999

Characterization of the 5' ends for polyadenylated RNAs synthesized during the replication of hepatitis delta virus.

The genome of hepatitis delta virus (HDV) is a 1,679-nucleotide (nt) single-stranded circular RNA that is predicted to fold into an unbranched rodlike structure. During replication, two complementary RNAs are also detected: an exact complement, referred to as the antigenome, and an 800-nt polyadenylated RNA that could act as the mRNA for the delta antigen. We used a 5' rapid amplification of cDNA ends procedure, followed by cloning and sequencing, to determine the 5' ends of the polyadenylated RNAs produced during HDV genome replication following initiation under different experimental conditions. The analyzed RNAs were from the liver of an infected woodchuck and from a liver cell line at 6 days after transfection with either an HDV cDNA or ribonucleoprotein (RNP) complexes assembled in vitro with HDV genomic RNA and purified recombinant small delta protein. In all three situations the 5' ends mapped specifically to nt 1630. In relationship to what is called the top end of the unbranched rodlike structure predicted for the genomic RNA template, this site is located 10 nt from the top, and in the middle of a 3-nt external bulge. Following transfection with RNP, such specific 5' ends could be detected as early as 24 h. We next constructed a series of mutants of this predicted bulge region and of an adjacent 6-bp stem and the top 5-nt loop. Some of these mutations decreased the ability of the genome to undergo antigenomic RNA synthesis and accumulation and/or altered the location of the detected 5' ends. The observed end located at nt 1630, and most of the novel 5' ends, were consistent with transcription initiation events that preferentially used a purine. The present studies do not prove that the detected 5' ends correspond to initiation sites and do not establish the hypothesis that there is a promoter element in the vicinity, but they do show that the location of the observed 5' ends could be controlled by nucleotide sequences at and around nt 1630.

5' Untranslated Regions↗
PubMed2000

Interactions between hepatitis delta virus proteins.

The 195- and 214-amino-acid (aa) forms of the delta protein (deltaAg-S and deltaAg-L, respectively) of hepatitis delta virus (HDV) differ only in the 19-aa C-terminal extension unique to deltaAg-L. deltaAg-S is needed for genome replication, while deltaAg-L is needed for particle assembly. These proteins share a region at aa 12 to 60, which mediates protein-protein interactions essential for HDV replication. H. Zuccola et al. (Structure 6:821-830, 1998) reported a crystal structure for a peptide spanning this region which demonstrates an antiparallel coiled-coil dimer interaction with the potential to form tetramers of dimers. Our studies tested whether predictions based on this structure could be extrapolated to conditions where the peptide was replaced by full-length deltaAg-S or deltaAg-L, and when the assays were not in vitro but in vivo. Nine amino acids that are conserved between several isolates of HDV and predicted to be important in multimerization were mutated to alanine on both deltaAg-S and deltaAg-L. We found that the predicted hierarchy of importance of these nine mutations correlated to a significant extent with the observed in vivo effects on the ability of these proteins to (i) support in trans the replication of the HDV genome when expressed on deltaAg-S and (ii) act as dominant-negative inhibitors of replication when expressed on deltaAg-L. We thus infer that these biological activities of deltaAg depend on ordered protein-protein interactions.

Amino Acid Sequence↗
PubMed2003

Eggplant latent viroid, the candidate type species for a new genus within the family Avsunviroidae (hammerhead viroids).

Viroids, small circular RNAs that replicate independently and in most cases incite diseases in plants, are classified into the families Pospiviroidae, composed of species with a central conserved region (CCR) and without hammerhead ribozymes, and Avsunviroidae, composed of three members lacking CCR but able to self-cleave in both polarity strands through hammerhead ribozymes. Here we report the biological and molecular properties of Eggplant latent viroid (ELVd). Purified circular ELVd induces symptomless infections when inoculated into eggplant seedlings. ELVd can be transmitted horizontally and through seed. Sequencing 10 complete cDNA clones showed that ELVd is a circular RNA of 332 to 335 nucleotides with high variability. This RNA can adopt a quasi-rod-like secondary structure of minimal free energy and alternative foldings that permit formation of stable hammerhead structures in plus and minus strands. The ribozymes are active in vitro and, most likely, in vivo. Considering the ELVd properties to be intermediate between those of the two genera of family Avsunviroidae, we propose ELVd as the type species of a third genus with the name ELAVIROID:

Base Sequence↗
PubMedDate not supplied

A Canadian isolate of hepatitis D (delta) virus.

Hepatitis D (delta) virus (HDV) is an infectious agent that propagates in hepatocytes only in the presence of hepatitis B virus, causing fulminant or chronic hepatitis with liver cirrhosis. HDV is a 36 nm particle that includes a circular RNA genome of 1.7 kilobases with an extensive internal complementary that allows it to fold into a rod-like structure. The relationships among genotypes, sequence variability, geographical distribution and disease severity of HDV remain unknown. Consequently, in the present study, the complete nucleotide sequence of an HDV isolated from a Canadian patient was determined. The viral RNA from serum was amplified using reverse transcription coupled to polymerase chain reaction amplification. The resulting complementary DNA was cloned and sequenced. Sequence analysis revealed that this new isolate contained 1672 nucleotides corresponding to genotype 1, which has a worldwide distribution. Sequencing of four independent clones revealed 17 substitutions, corresponding to an overall sequence variability of 1%. Surprisingly, seven mutations were found in the 48-nucleotide region located between the two highly conserved self-catalytic motifs. This is the first demonstration that many substitutions are identified in this region of HDV, and prompts the present authors to define it as a hypervariable region.

Base Sequence↗
PubMed2024

RNA-seq reveals differentially expressed lncRNAs and circRNAs and their associated functional network in HTR-8/Svneo cells under hypoxic conditions.

Placental hypoxia is hazardous to maternal health as well as fetal growth and development. Preeclampsia and intrauterine growth restriction are common pregnancy problems, and one of the causes is placental hypoxia. Placental hypoxia is linked to a number of pregnancy illnessesv. To investigate their potential function in anoxic circumstances, we mimicked the anoxic environment of HTR-8/Svneo cells and performed lncRNA and circRNA studies on anoxic HTR-8/Svneo cells using high-throughput RNA sequencing. The miRNA target genes were predicted by integrating the aberrant expression of miRNAs in the placenta of preeclampsia and intrauterine growth restriction, and a ceRNA network map was developed to conduct a complete transcriptomic and bioinformatics investigation of circRNAs and lncRNAs. The signaling pathways in which the genes were primarily engaged were predicted using GO and KEGG analyses. To propose a novel explanation for trophoblastic organism failure caused by lncRNAs and circRNAs in an anoxic environment.

Humans↗
PubMed1995

Expression of Sry, the mouse sex determining gene.

In the mouse, Sry is expressed by germ cells in the adult testis and by somatic cells in the genital ridge. Transcripts in the former exist as circular RNA molecules of 1.23 kb, which are unlikely to be efficiently translated. We have used RNase protection to map the extent of the less abundant Sry transcript in the developing gonad. We demonstrate that it is a linear mRNA derived from a single exon. This begins in the unique region 5' of the protein coding region and extends several kilobases into the 3' arm of the large inverted repeat which bounds the Sry genomic locus. Knowledge of this transcript, which is very different from that of the human SRY gene, allows us to predict its protein product and reveals several features which may be involved in translational control. Our data is also consistent with there being two promoters for the Sry gene, a proximal one that gives functional transcripts in the genital ridge and a distal promoter used in germ cells in the adult testis. As RNase protection is a quantitative technique, a detailed timecourse of Sry expression was carried out using accurately staged samples. Sry transcripts are first detectable just after 10.5 days post coitum, they reach a peak at 11.5 days and then decline sharply so that none are detected 24 hours later. This was compared with anti-Müllerian hormone gene expression, an early marker of Sertoli cells and the first known downstream gene of Sry. Amh expression begins 20 hours after the onset of Sry expression at a time when Sry transcripts are at their peak. While this result does not prove a direct interaction between the two genes, it defines the critical period during which Sry must act to initiate Sertoli cell differentiation.

Amino Acid Sequence↗
PubMed1999

Linear SRY transcript in equine testis.

Employing a combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques, the complete coding sequence of cDNA for the equine SRY gene was determined. We also attempted to make clear whether the equine SRY gene transcript is expressed in the adult testis, and whether the type of transcript is expressed as linear or circular RNA. As a result, in total a 1420 bp cDNA sequence was determined. Accomplishment of 3' RACE infers that equine SRY gene was expressed as a linear RNA transcript in testicular tissue just after puberty, in contrast to the situation in mice.

Amino Acid Sequence↗
PubMedDate not supplied

Viroids with hammerhead ribozymes: some unique structural and functional aspects with respect to other members of the group.

Viroids, subviral pathogens of plants, are composed of a single-stranded circular RNA of 246-399 nucleotides. Within the 27 viroids sequenced, avocado sunblotch, peach latent mosaic and chrysanthemum chlorotic mottle viroids (ASBVd, PLMVd and CChMVd, respectively) can form hammerhead structures in both of their polarity strands. These ribozymes mediate self-cleavage of the oligomeric RNAs generated in the replication through a rolling circle mechanism, whose two other steps are catalyzed by an RNA polymerase and an RNA ligase. ASBVd, and presumably PLMVd and CChMVd, replicate and accumulate in the chloroplast, whereas typical viroids replicate and accumulate in the nucleus. PLMVd and CChMVd do not adopt a rod-like or quasi rod-like secondary structure as typical viroids do but have a highly branched conformation. A pathogenicity determinant has been mapped in a defined region of the CChMVd molecule.

Base Sequence↗
PubMedDate not supplied

[Hepatitis D].

The hepatitis D virus (HDV), also called delta virus, is a small circular RNA virus. The HDV is dependent on the hepatitis B virus (HBV) and can cause infection in normal individuals with hepatitis B or yet, superinfect chronic HBV carriers. Three genotypes have already been cloned and sequenced. Infection with HDV has a worldwide distribution and a high HDV endemicity has been documented in the western Amazon region, in Brazil. It has been estimated that 18 million people are infected with this virus amongst the 350 million carriers of the HBV around the world. The HDV transmission and risk factors for infection are similar to those for HBV infection. The diagnosis is based on the immunohistological identification of HDAg in the liver and detection of IgM and IgG anti-HD in serum using RIA or EIA. The clinical course of hepatitis D is variable. Fulminant disease occurs more commonly in hepatitis B and D than in other forms of acute viral hepatitis. Chronic HDV infection is usually associated with severe histological changes in the liver and with a rapidly progressive course, that can lead to cirrhosis, liver failure and death. Treatment of chronic hepatitis D is currently unsatisfactory and interferon alpha is the only agent found to have some effect on the course of chronic hepatitis. Orthotopic liver transplantation is indicated for terminal cases of cirrhosis. Prophylaxis for HDV infection is possible by vaccination against the hepatitis B virus.

Hepatitis D↗
PubMed1992

The main five types of viral hepatitis: an alphabetical update.

The rapidly increasing knowledge in the field of viral hepatitis warrants regular updates. Clinical studies with new hepatitis A vaccines have shown that they are safe, well-tolerated, and effective. Several reports on hepatitis B virus (HBV) variants have appeared. Surface antigen mutants may have an important influence on vaccine prophylaxis because existing vaccines may not protect against infection with these variants. Hepatitis D virus is a circular RNA virus that requires the presence of HBV for successful infection. The requirements for the dual expression of these viruses are unknown and their relation is complex. Hepatitis C virus (HCV) is a RNA virus that has homology with the flaviviridae. This is a rather common agent in most populations studied and often causes chronic infection but little is known about its spread. Hepatitis E virus is a RNA virus which is usually spread by contaminated water in developing countries. The disease causes high mortality in pregnant women. The existence of further viral hepatitis agents have been suggested but hard data confirming this is so far lacking.

Animals↗
PubMed1997

The latency associated transcripts (LAT) of herpes simplex virus: still no end in sight.

The herpes simplex virus latency associated transcripts (LAT) are the only viral gene products that accumulate to a high concentration in the trigeminal ganglia (TG) of latently infected animals. Their abundance is particularly surprising, since they are thought to be the introns of a larger, approximately 8.3 kb precursor. LAT are not linear molecules. Therefore they are either a circle or a lariat that is not debranched. This structure could explain their unusual high stability. Moreover, the functional potential of stable, nuclear RNA has been demonstrated in other biological systems and could offer a clue as to the mechanism of action of LAT. Therefore, the non-linear nature of LAT and functional implications mean that both literally and figuratively, there is no end in sight for this unusual molecule.

Animals↗
PubMed1987

Purification of a RNA debranching activity from HeLa cells.

The splicing of messenger RNA precursors (pre-mRNA) of eukaryotic cells involves the formation of a branched RNA intermediate known as a RNA lariat. This structure is formed in the first step of the reaction when a cleavage at the 5' splice site generates the 5' exon and a RNA species containing the intron and 3' exon in which the phosphate moiety at the 5' end of the intron is forming a 2'-5' phosphodiester bond with the 2'-hydroxyl moiety of a specific adenine residue near the 3' end of the intron forming a RNA branch with the following structure: -pA2'-pX-3'-pZ-. We have purified a debranching activity approximately 700-fold from the cytosolic fraction of HeLa cells. This activity catalyzes the hydrolysis of the 2'-5' phosphodiester bond of branched RNA structures yielding a 5'-phosphate end and a 2'-hydroxyl group at the branch attachment site. The activity possessed a sedimentation coefficient of 3.5 S. The reaction catalyzed by the purified fraction requires a divalent cation and is optimal at pH 7.0. The purified activity can efficiently hydrolyze triester trinucleotide structures (pY2'-pX-3'-pZ-) prepared by digestion of RNA lariats with nuclease P1. In contrast, a 2' phosphate monoester product (-pG2'-p 3'-pC-), formed by the wheat germ RNA ligase, was not attacked.

Cations, Divalent↗
PubMed1987

Delta agent hepatitis.

Since its chance discovery a mere decade ago, the delta agent has been characterized as a novel pathogen that poses the risk of developing into a scourge of modern times. With its unique single-stranded circular RNA, exclusive dependence on HBV for its replication, and characteristic ability to suppress hepatitis B synthesis, the delta agent has emerged as an important global cause of fulminant hepatitis and progressive liver disease. Persons at greatest risk of delta infection are those living in endemic areas and HBsAg-carrier parenteral drug abusers, hemophiliacs, hemodialysis patients, and homosexual men in nonendemic areas. Widespread dissemination appears possible. There is as yet no known effective treatment for established delta infection, and, for the present, clinicians must concentrate on preventing HDV infection by incorporating vigorous use of the hepatitis B vaccine into strategies for preventing HBV infection.

Hepatitis D↗
PubMed1994

Multiple guide RNAs for identical editing of Trypanosoma brucei apocytochrome b mRNA have an unusual minicircle location and are developmentally regulated.

We identified four different guide RNAs (gRNAs) that specify identical editing of Trypanosoma brucei apocytochrome b (CYb) mRNA, which indicates gRNA redundancy in T. brucei. All four gRNAs appear functional since they occur in chimeras, some of which contain an interesting gRNA 3' "extension." The gRNAs are encoded in different minicircles, rather than maxicircles as in other species. However, these gRNA genes are not between 18-base pair repeats as are the other minicircle gRNA genes in T. brucei. The three minicircles cloned contain the same gRNA genes, one of which is substantially diverged, all in the same order, indicating that they are related. CYb gRNA is less abundant in procyclic than bloodstream forms. Procyclic forms contain abundant edited CYb mRNA unlike bloodstream forms thus suggesting that CYb mRNA editing may be regulated at the level of gRNA utilization.

Animals↗