In vivo expression of the nucleolar group I intron-encoded I-dirI homing endonuclease involves the removal of a spliceosomal intron.
The Didymium iridis DiSSU1 intron is located in the nuclear SSU rDNA and has an unusual twin-ribozyme organization. One of the ribozymes (DiGIR2) catalyses intron excision and exon ligation. The other ribozyme (DiGIR1), which along with the endonuclease-encoding I-DirI open reading frame (ORF) is inserted in DiGIR2, carries out hydrolysis at internal processing sites (IPS1 and IPS2) located at its 3' end. Examination of the in vivo expression of DiSSU1 shows that after excision, DiSSU1 is matured further into the I-DirI mRNA by internal DiGIR1-catalysed cleavage upstream of the ORF 5' end, as well as truncation and polyadenylation downstream of the ORF 3' end. A spliceosomal intron, the first to be reported within a group I intron and the rDNA, is removed before the I-DirI mRNA associates with the polysomes. Taken together, our results imply that DiSSU1 uses a unique combination of intron-supplied ribozyme activity and adaptation to the general RNA polymerase II pathway of mRNA expression to allow a protein to be produced from the RNA polymerase I-transcribed rDNA. (+info)
Nucleotide sequences and taxonomy of satsuma dwarf virus.
The nucleotide sequences of genomic RNA1 (6795 nt) and RNA2 (5345 nt) of satsuma dwarf virus (SDV), a tentative member of the genus Nepovirus, were determined. The deduced genome organization of SDV showed similarities to the organization in como-, faba- and nepoviruses. There is extensive amino acid sequence similarity in the N-terminal regions of the proteins encoded by RNA1 and RNA2, as reported previously only for tomato ringspot nepovirus. However, unlike definitive nepoviruses, which have a single coat protein, SDV has two coat proteins. SDV RNA2 does not contain the long (> 1300 nt) 3' non-coding region characteristic of some nepoviruses. Phylogenetic analysis of SDV RNA polymerase placed SDV apart from como-, faba- and nepoviruses. These unique features suggest that SDV is distinct from the Comovirus, Fabavirus and Nepovirus genera, and needs to be separated into a new genus, probably within the family Comoviridae. (+info)
Degradation of 1,2-dibromoethane by Mycobacterium sp. strain GP1.
The newly isolated bacterial strain GP1 can utilize 1, 2-dibromoethane as the sole carbon and energy source. On the basis of 16S rRNA gene sequence analysis, the organism was identified as a member of the subgroup which contains the fast-growing mycobacteria. The first step in 1,2-dibromoethane metabolism is catalyzed by a hydrolytic haloalkane dehalogenase. The resulting 2-bromoethanol is rapidly converted to ethylene oxide by a haloalcohol dehalogenase, in this way preventing the accumulation of 2-bromoethanol and 2-bromoacetaldehyde as toxic intermediates. Ethylene oxide can serve as a growth substrate for strain GP1, but the pathway(s) by which it is further metabolized is still unclear. Strain GP1 can also utilize 1-chloropropane, 1-bromopropane, 2-bromoethanol, and 2-chloroethanol as growth substrates. 2-Chloroethanol and 2-bromoethanol are metabolized via ethylene oxide, which for both haloalcohols is a novel way to remove the halide without going through the corresponding acetaldehyde intermediate. The haloalkane dehalogenase gene was cloned and sequenced. The dehalogenase (DhaAf) encoded by this gene is identical to the haloalkane dehalogenase (DhaA) of Rhodococcus rhodochrous NCIMB 13064, except for three amino acid substitutions and a 14-amino-acid extension at the C terminus. Alignments of the complete dehalogenase gene region of strain GP1 with DNA sequences in different databases showed that a large part of a dhaA gene region, which is also present in R. rhodochrous NCIMB 13064, was fused to a fragment of a haloalcohol dehalogenase gene that was identical to the last 42 nucleotides of the hheB gene found in Corynebacterium sp. strain N-1074. (+info)
Salmon pancreas disease virus, an alphavirus infecting farmed Atlantic salmon, Salmo salar L.
A 5.2-kb region at the 3' terminus of the salmon pancreas disease virus (SPDV) RNA genome has been cloned and sequenced. The nucleotide and predicted amino acid sequences show that SPDV shares considerable organizational and sequence identity to members of the genus alphavirus within the family Togaviridae. The SPDV structural proteins encoded by the 5.2-kb region contain a number of unique features when compared to other sequenced alphaviruses. Based on cleavage site homologies, the predicted sizes of the SPDV envelope glycoproteins E2 (438 aa) and E1 (461 aa) are larger than those of other alphaviruses, while the predicted size of the alphavirus 6K protein is 3.2 K (32 aa) in SPDV. The E2 and E1 proteins each carry one putative N-linked glycosylation site, with the site in E1 being found at a unique position. From amino acid sequence comparisons of the SPDV structural region with sequenced alphaviruses overall homology is uniform, ranging from 32 to 33%. While nucleotide sequence analysis of the 26S RNA junction region shows that SPDV is similar to other alphaviruses, analysis of the 3'-nontranslated region reveals that SPDV shows divergence in this region. (+info)
Sequence and structural elements at the 3' terminus of bovine viral diarrhea virus genomic RNA: functional role during RNA replication.
Bovine viral diarrhea virus (BVDV), a member of the genus Pestivirus in the family Flaviviridae, has a positive-stranded RNA genome consisting of a single open reading frame and untranslated regions (UTRs) at the 5' and 3' ends. Computer modeling suggested the 3' UTR comprised single-stranded regions as well as stem-loop structures-features that were suspected of being essentially implicated in the viral RNA replication pathway. Employing a subgenomic BVDV RNA (DI9c) that was shown to function as an autonomous RNA replicon (S.-E. Behrens, C. W. Grassmann, H. J. Thiel, G. Meyers, and N. Tautz, J. Virol. 72:2364-2372, 1998) the goal of this study was to determine the RNA secondary structure of the 3' UTR by experimental means and to investigate the significance of defined RNA motifs for the RNA replication pathway. Enzymatic and chemical structure probing revealed mainly the conserved terminal part (termed 3'C) of the DI9c 3' UTR containing distinctive RNA motifs, i.e., a stable stem-loop, SL I, near the RNA 3' terminus and a considerably less stable stem-loop, SL II, that forms the 5' portion of 3'C. SL I and SL II are separated by a long single-stranded intervening sequence, denoted SS. The 3'-terminal four C residues of the viral RNA were confirmed to be single stranded as well. Other intramolecular interactions, e.g., with upstream DI9c RNA sequences, were not detected under the experimental conditions used. Mutagenesis of the DI9c RNA demonstrated that the SL I and SS motifs do indeed play essential roles during RNA replication. Abolition of RNA stems, which ought to maintain the overall folding of SL I, as well as substitution of certain single-stranded nucleotides located in the SS region or SL I loop region, gave rise to DI9c derivatives unable to replicate. Conversely, SL I stems comprising compensatory base exchanges turned out to support replication, but mostly to a lower degree than the original structure. Surprisingly, replacement of a number of residues, although they were previously defined as constituents of a highly conserved stretch of sequence of the SS motif, had little effect on the replication ability of DI9c. In summary, these results indicate that RNA structure as well as sequence elements harbored within the 3'C region of the BVDV 3' UTR create a common cis-acting element of the replication process. The data further point at possible interaction sites of host and/or viral proteins and thus provide valuable information for future experiments intended to identify and characterize these factors. (+info)
In vitro infection of human peripheral blood mononuclear cells by GB virus C/Hepatitis G virus.
GB virus C (GBV-C), also known as hepatitis G virus, is a recently discovered flavivirus-like RNA agent with unclear pathogenic implications. To investigate whether human peripheral blood mononuclear cells (PBMC) are susceptible to in vitro GBV-C infection, we have incubated PBMC from four healthy blood donors with a human GBV-C RNA-positive serum. By means of (i) strand-specific reverse transcription-PCR, cloning, and sequencing; (ii) sucrose ultracentrifugation and RNase sensitivity assays; (iii) fluorescent in situ hybridization; and (iv) Western blot analysis, it has been demonstrated that GBV-C is able to infect in vitro cells and replicate for as long as 30 days under the conditions developed in our cell culture system. The concentration of GBV-C RNA increased during the second and third weeks of culture. The titers of the genomic strand were 10 times higher than the titers of the antigenomic strand. In addition, the same predominant GBV-C sequence was found in all PBMC cultures and in the in vivo-GBV-C-infected PBMC isolated from the donor of the inoculum. GBV-C-specific fluorescent in situ hybridization signals were confined to the cytoplasm of cells at different times during the culture period. Finally, evidence obtained by sucrose ultracentrifugation, RNase sensitivity assays, and Western blot analysis of the culture supernatants suggests that viral particles are released from in vitro-GBV-C-infected PBMC. In conclusion, our study has demonstrated, for the first time, GBV-C replication in human lymphoid cells under experimental in vitro infection conditions. (+info)
Comparative sequence analysis of tmRNA.
Minimal secondary structures of the bacterial and plastid tmRNAs were derived by comparative analyses of 50 aligned tmRNA sequences. The structures include 12 helices and four pseudoknots and are refinements of earlier versions, but include only those base pairs for which there is comparative evidence. Described are the conserved and variable features of the tmRNAs from a wide phylogenetic spectrum, the structural properties specific to the bacterial subgroups and preliminary 3-dimensional models from the pseudoknotted regions. (+info)
Attenuated virulence of pleconaril-resistant coxsackievirus B3 variants.
Pleconaril (VP 63843) is a novel orally bioavailable small molecule with broad antipicornavirus (enterovirus and rhinovirus) activity. Ten independently derived pleconaril-resistant variants of coxsackievirus B3 were isolated from cell culture. The molecular basis of drug resistance and the biologic properties of the drug-resistant viruses were investigated. RNA sequence analysis revealed amino acid changes in the drug-binding pocket of the resistant variants. Thermal stability studies showed the drug-resistant viruses to be significantly less stable than wild type virus. When evaluated in a murine model in which wild type virus infection is 100% lethal, the drug-resistant viruses showed attenuated virulence with both reduced mortality and delayed time to death. Virus titers in heart and spleen were dramatically lower in drug-resistant virus-infected mice than in wild type virus-infected animals. The study results indicate that pleconaril-resistant virus variants are attenuated and significantly less virulent than drug-sensitive wild type virus. (+info)