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At least 181 records · Page 10Linked to original sources
PubMed2005

Dominant optic atrophy caused by a novel OPA1 splice site mutation (IVS20+1G-->A) associated with intron retention.

Dominant optic atrophy (DOA) is the most common form of inherited primary optic neuropathy. The purpose of the current study was to report a novel OPA1 splice site mutation and investigate the impact of the mutation on pre-mRNA splicing in a female proband and her father diagnosed with DOA. We evaluated visual acuity, retinal fundi and kinetic visual fields. Color vision phenotypes were determined using the Farnsworth Panel D-15 and the Farnsworth-Munsell 100-hue tests. All 28 coding exons of the OPA1 gene were analyzed with polymerase chain reaction (PCR) amplification and direct sequencing. Total RNA extraction from white blood cells followed by reverse transcription-PCR (RT-PCR) was performed. We identified a novel heterozygous G to A mutation at position +1 of intron 20 (g.IVS20+1G-->A) in both patients. RT-PCR analysis revealed that the first 25 bp from intron 20 plus exon 20 were spliced onto exon 21. No difference in expression of mutant and wild-type transcripts was found within the linear range of amplification. Clinically, both patients exhibited reduced visual acuities, pallor of optic discs, decreased sensitivities of central visual fields and blue-yellow color vision defects. Previously, only one mechanism (skipping of exon) of pre-mRNA splicing defects has been reported among OPA1 splice site mutations. Our study demonstrates that the mechanism of intron retention is a novel type of pre-mRNA splicing defects. The mutant transcript with a premature termination codon is likely to encode a truncated protein, due to a translational frameshift (V672fsX675), that lacks 289 amino acids of the C-terminal end. Therefore, it is suggested that haploinsufficiency underlies DOA in the patients. However, we could not exclude the possibility that the truncated protein has a dominant negative activity because the mutant transcript is insusceptible to nonsense-mediated mRNA decay.

Adult↗
PubMed2005

Synthetic hammerhead ribozymes as therapeutic tools to control disease genes.

Ribozymes are RNA molecules that have the ability to catalyse the cleavage and formation of covalent bonds in RNA strands at specific sites. The "hammerhead" motif, approximately 30-nucleotide long, is the smallest endonucleolytic cis-acting ribozyme structure found in natural circular RNAs of some plant viroids. Hammerhead ribozymes became appealing when it was shown that it is possible to produce trans-acting ribozymes directed against RNA sequences of interest. Since then, gene-tailored ribozymes have been designed, produced and given to cells to knock down the expression of specific genes. At present, this technology has advanced so much that many hammerhead ribozymes are being used in clinical trials. With this work we would provide some guidelines to design efficient trans-acting hammerhead ribozymes as well as review the recent results obtained with them as gene therapy tools.

Animals↗
PubMed2000

Genotypic and phenotypic nevirapine resistance correlates with virological failure during salvage therapy including abacavir and nevirapine.

OBJECTIVE: To study the development of resistance during 8 weeks of salvage therapy with abacavir and nevirapine in combination with other reverse transcriptase inhibitors (RTIs) and protease inhibitors (PIs). METHODS: Samples obtained at baseline and after 8 weeks of therapy from 16 heavily pretreated patients were analysed for genotypic and phenotypic resistance. Genotypic resistance was analysed in cell-associated DNA and plasma HIV-RNA using direct sequencing. Phenotypic resistance was analysed in a PBMC-based assay and in a recombinant virus assay. Plasma viral load was measured at baseline and after 2, 4 and 8 weeks of therapy. RESULTS: The majority of patients was genotypically and phenotypically resistant to lamivudine, abacavir, zidovudine and PIs, whereas 50% of the patients showed resistance to nevirapine at baseline in at least one of the methods used. After 8 weeks of salvage therapy, no additional development of resistance against nucleoside reverse transcriptase inhibitors and PIs could be detected. However, the amount of patients resistant to nevirapine increased to 83%. When the patients were divided into two groups according to baseline resistance against nevirapine, a significantly higher transient reduction in viral load was observed in patients with nevirapine-sensitive HIV at baseline compared to patients with resistant HIV at baseline. CONCLUSION: The transient effect of salvage therapy including abacavir and nevirapine was due to the effect of nevirapine. The lack of effect of abacavir was most likely due to cross-resistance between abacavir and lamivudine/zidovudine used in previous treatment.

Anti-HIV Agents↗
PubMed1993

Phylogenetic relationships of Baikalian species of Prorhynchidae turbellarian worms as inferred by partial 18S rRNA gene sequence comparisons (preliminary report).

The polymerase chain reaction (PCR) and direct sequencing of RNA genes were applied for phylogenetic relationship analysis among Turbellaria (flatworms). Representatives of 10 species of the genus Geocentrophora (Prorhynchidae family) were used to establish homology between nucleotides in the 5'-end portion of the 18S rRNA gene sequences. Sequence data were subdivided into 3 subsets--unpaired and basepaired (according to rRNA secondary structure) and transversion, including only transversion substitutions--that were independently analyzed to construct phylogenetic trees. Distance matrix and maximum parsimony approaches were applied to infer phylogenies. Trees were examined in terms of morphological taxonomy. The DNA sequences support the taxonomy and systematics of Baikalian flatworms. The hypothesis of Geocentrophora origin in Baikal is discussed.

Animals↗
PubMed1996

Structural and mechanistic relationships between nucleic acid polymerases.

A superfamily of nucleic acid polymerases that includes the pol I and pol alpha classes of DNA-directed DNA polymerases, mitochondrial and phage DNA-directed RNA polymerases, and most RNA-directed polymerases may be defined on the basis of the occurrence of conserved sequence motifs and tertiary structure similarities between HIV-1 reverse transcriptase, DNA polymerase I and T7 RNA polymerase. Although sequence or structural similarities do not yet justify inclusion of the multi-subunit DNA-directed RNA polymerases in this superfamily, mechanistic similarities suggest a deep relationship between these and the simpler T7-like RNA polymerases.

Amino Acid Sequence↗
PubMed1998

Recognition sites of 3'-OH group by T7 RNA polymerase and its application to transcriptional sequencing.

When analyzing the elongation mechanisms in T7 RNA polymerase (T7 RNAP)by using site-directed mutagenesis and a protein expression system, we identified the recognition sites of the rNTP 3'-OH group in T7 RNAP. On the basis of three-dimensional crystal structure analysis, we selected and analyzed six candidate sites interacting with the 3'-OH group of rNTP in T7 RNAP. We found that the Phe-644 and Phe-667 sites are responsible for the high selectivity of T7 RNAP for rNTPs. Also, we constructed the protein mutations of these residues, F644Y and F667Y, which display a >200-fold higher affinity than the wild type for 3'-dNTPs. These findings indicate that the phenylalanine residues of 644 and 667 specifically interact with the 3'-OH group. Thus, these mutants, F644Y and F667Y, with incorporation of 3'-dNTP terminators, which is similar to native rNTPs, can offer low backgrounds and equal intensities of the sequencing ladders in our method, called "transcriptional sequencing. "

Amino Acid Sequence↗
PubMed1990

Origin and evolution of retroelements based upon their reverse transcriptase sequences.

To study the evolutionary relationship of reverse transcriptase (RT) containing genetic elements, a phylogenetic tree of 82 retroelements from animals, plants, protozoans and bacteria was constructed. The tree was based on seven amino acid domains totalling 178 residues identified in all RTs. We have also identified these seven domains in the RNA-directed RNA polymerases from various plus-strand RNA viruses. The sequence similarity of these RNA polymerases to RT suggests that these two enzymes evolved from a common ancestor, and thus RNA polymerase can be used as an outgroup to root the RT tree. A comparison of the genetic organization of the various RT containing elements and their position on the tree allows several inferences concerning the origin and evolution of these elements. The most probable ancestor of current retroelements was a retrotransposable element with both gag-like and pol-like genes. On one major branch of the tree, organelle and bacterial sequences (e.g. group II introns and bacterial msDNA) appear to have captured the RT sequences from retrotransposons which lack long terminal repeats (LTRs). On the other major branch, acquisition of LTRs gave rise to two distinct groups of LTR retrotransposons and three groups of viruses: retroviruses, hepadnaviruses and caulimoviruses.

Amino Acid Sequence↗
PubMed1987

Plastid, nuclear and reverse transcriptase sequences in the mitochondrial genome of Oenothera: is genetic information transferred between organelles via RNA?

We describe an open reading frame (ORF) with high homology to reverse transcriptase in the mitochondrial genome of Oenothera. This ORF displays all the characteristics of an active plant mitochondrial gene with a possible ribosome binding site and 39% T in the third codon position. It is located between a sequence fragment from the plastid genome and one of nuclear origin downstream from the gene encoding subunit 5 of the NADH dehydrogenase. The nuclear derived sequence consists of 528 nucleotides from the small ribosomal RNA and contains an expansion segment unique to nuclear rRNAs. The plastid sequence contains part of the ribosomal protein S4 and the complete tRNA(Ser). The observation that only transcribed sequences have been found i more than one subcellular compartment in higher plants suggests that interorganellar transfer of genetic information may occur via RNA and subsequent local reverse transcription and genomic integration.

Base Sequence↗
PubMed1996

Identification of cis-acting sequences that control nanos RNA localization.

The generation of anterior-posterior polarity during development in Drosophila requires the localization of determinant molecules to the anterior and posterior poles of the embryo. Localization of the maternally synthesized nanos RNA to the posterior pole of the embryo is essential to provide a source for a gradient of Nanos protein that directs abdomen formation. nanos RNA localization occurs during oogenesis and requires the function of at least nine genes. cis-acting sequences that direct nanos RNA localization lie within the nanos 3'UTR. In this analysis, we have used nanos 3'UTR deletion mutants to define the localization signal more precisely. Our results indicate that the nanos RNA localization signal is large and complex and that targeting of nanos RNA may be achieved by the combined effects of multiple, partially independent sequences. This idea is supported by evolutionary conservation, both in sequence and in function, of the nanos 3'UTRs of Drosophila melanogaster and Drosophila virilis.

Animals↗
PubMed1993

The phage RNA polymerases are related to DNA polymerases and reverse transcriptases.

The single subunit DNA-dependent RNA polymerase (RNAP) that is encoded by bacteriophage T7 is the prototype of a class of relatively simple RNAPs that includes the RNAPs of the related phages T3 and SP6, as well as the mitochondrial RNAPs. The T7 enzyme has been crystallized, and recent genetic and biochemical analyses have facilitated an interpretation of this structure. A growing body of evidence suggests that the phage-like RNAPs are related to other nucleotide polymerases such as DNA polymerases, RNA-dependent RNA polymerases, and reverse transcriptases. In this work, we review information concerning the structure and function of T7 RNAP, and evidence in support of its assignment to a broader class of nucleotide polymerases.

Amino Acid Sequence↗
PubMed1997

Minimal templates directing accurate initiation of subgenomic RNA synthesis in vitro by the brome mosaic virus RNA-dependent RNA polymerase.

Short regions of (-)-strand brome mosaic virus (BMV) RNA3, proscripts, were shown to direct accurate in vitro synthesis of (+)-strand subgenomic RNA by the BMV RNA-dependent RNA polymerase (RdRp), facilitating characterization of the sequences and/or structures directing subgenomic RNA synthesis. Proscripts retaining fewer than 8-nt of an 18-nt polyuridylate tract located just upstream of the core promoter sequence (French R, Ahlquist P, 1988, J Virol 62:2411-2420; Marsh LE, Dreher TW, Hall TC, 1988, Nucleic Acids Res 16:981-995) directed dramatically less synthesis of 26-nt or longer products. Original levels of RNA synthesis were not restored by replacement of the 3' polyuridylate tract with polyadenylate, polycytidylate, or polyguanylate tracts, or by movement of the polyuridylate tract to the 5' end of the proscript. The polyuridylate tract presumably binds some component(s) required for RdRp activity because the addition of poly(U) [but not poly(C)] RNA to RdRp reactions decreased RNA synthesis significantly. Quite surprisingly, deletions of the polyuridylate tract in proscripts directing synthesis of 24-nt or shorter products had little or no detrimental effect on subgenomic RNA synthesis, correlated with their inability to form a computer-predicted stem-loop present in longer proscripts requiring the polyuridylate tract. Successive 3' and 5' deletions demonstrated that the minimal elements required for accurate initiation of subgenomic RNA synthesis are within a proscript of 22-nt.

Bromovirus↗
PubMed1999

RNA sequence and secondary structural determinants in a minimal viral promoter that directs replicase recognition and initiation of genomic plus-strand RNA synthesis.

Viral RNA replication provides a useful system to study the structure and function of RNAs and the mechanism of RNA synthesis from RNA templates. Previously we demonstrated that a 27 nt RNA from brome mosaic virus (BMV) can direct correct initiation of genomic plus-strand RNA synthesis by the BMV replicase. In this study, using biochemical, nuclear magnetic resonance, and thermodynamic analyses, we determined that the secondary structure of this 27 nt RNA can be significantly altered and retain the ability to direct RNA synthesis. In contrast, we find that position-specific changes in the RNA sequence will affect replicase recognition, modulate the polymerization process, and contribute to the differential accumulation of viral RNAs. These functional results are in agreement with the phylogenetic analysis of BMV and related viral sequences and suggest that a similar mechanism of RNA synthesis takes place for members of the alphavirus superfamily.

Base Sequence↗
PubMed2001

Occurrence of genetic drift and founder effect during quasispecies evolution of the VP2 and NS3/NS3A genes of bluetongue virus upon passage between sheep, cattle, and Culicoides sonorensis.

Bluetongue virus (BTV) is the cause of an insect-transmitted virus infection of ruminants that occurs throughout much of the world. Individual gene segments differ between field strains of BTV; thus, we hypothesized that key viral genes undergo genetic drift during alternating passage of BTV in its ruminant and insect hosts. To test this hypothesis, variation in the consensus sequence and quasispecies heterogeneity of the VP2 and NS3/NS3A genes of a plaque-purified strain of BTV serotype 10 was determined during alternating infection of vector Culicoides sonorensis and a sheep and calf. Consensus sequences were determined after reverse transcriptase-nested PCR amplification of viral RNA directly from ruminant blood and homogenized insects, and quasispecies heterogeneity was determined by the sequencing of clones derived from directly amplified viral RNA. Comparison of these sequences to those of the original BTV inoculum used to initiate the cycle of BTV infection demonstrated, for the first time, that individual BTV gene segments evolve independently of one another by genetic drift in a host-specific fashion, generating quasispecies populations in both ruminant and insect hosts. Furthermore, a unique viral variant was randomly ingested by C. sonorensis insects that fed on a sheep with low-titer viremia, thereby fixing a novel genotype by founder effect. Thus, we conclude that genetic drift and founder effect contribute to diversification of individual gene segments of field strains of BTV.

Animals↗
PubMed2001

Molecular characterization of human enteroviruses in clinical samples: comparison between VP2, VP1, and RNA polymerase regions using RT nested PCR assays and direct sequencing of products.

Three nested RT-PCR assays were developed to permit sensitive typing of enteroviruses directly from clinical samples. These assays amplified short fragments from different genomic regions codifying for three proteins: VP2, VP1, and RNA polymerase. Given that enteroviruses have a high rate of degeneration within target codons among serotypes, the primers used consisted of mixed base and deoxyinosine residues. These techniques detected at 0.03-0.003 TCID50 of prototype Poliovirus 1 and Echovirus 30. They were used to characterize the enteroviral RNA detected in 18 CSF, stool, and throw swab samples and in 8 enterovirus isolates from patients with several syndromes. Phylogenetic analysis in each independent sequenced region grouped the enterovirus into four clusters, enabling genetic classification. A comparative study was performed among the 26 sequences obtained after direct sequencing of products with those available in the nucleotide databases. The efficiency of each assay for enterovirus identification was evaluated by both distance (Clustal) and similarity (M-NW) indices. Comparative results obtained independently in the three regions showed the highest yield of correlation between nucleotide sequences of all prototype serotypes and the analyzed genotypes in the VP1 region (26/26, 100% Clustal; 22/26, 85% M-NW). Conversely, the VP2 region failed to identify some of the circulating enteroviruses (17/26, 65% Clustal; 16/26, 62% M-NW). Using the RNA polymerase region, sequences from samples and isolates were associated with prototype strains whenever these were available (20/21, 95% Clustal; 12/21, 57% M-NW). These assays were useful for molecular identification of enterovirus directly from samples even when isolation was not possible.

Capsid↗
PubMed2000

RNA-directed DNA methylation.

RNA-DNA interactions can serve as a signal that triggers de novo DNA methylation in plants. As yet, this RNA-directed DNA methylation mechanism merely targets transgenes, but it appears likely that methylation of some endogenous sequences is also directed by RNA. RNA-directed methylation of cytosine residues specifically occurs along the DNA regions that are complementary to the directing RNA pointing to the formation of a putative RNA-DNA duplex. Dense methylation patterns and the methylation of cytosine residues at symmetric and asymmetric sites are detectable on both DNA strands within these regions. Methylation progressively decreases in the sequences adjacent to the putative RNA-DNA duplex. The extreme sensitivity of RNA-directed DNA methylation was demonstrated by analysing a short 30 bp DNA region that was complementary to the targeting RNA. Association of RNA-directed DNA methylation with homology-dependent gene silencing indicated that the methylation-directing RNA molecules may be double-stranded or may contain double-stranded regions. Whereas the function of DNA methylation in transcriptional gene silencing is nearly understood, its role in post-transcriptional gene silencing is still under discussion. In mammals, X-chromosome inactivation and genomic imprinting are associated with DNA methylation but how methylation is initiated is unclear. The observation of a correlation between specific antisense RNAs and transcriptional and post-transcriptional gene silencing may indicate that RNA-directed DNA methylation is involved in epigenetic gene regulation throughout eukaryotes.

Base Sequence↗
PubMed2002

Sequence requirements for viral RNA replication and VPg uridylylation directed by the internal cis-acting replication element (cre) of human rhinovirus type 14.

Until recently, the cis-acting signals required for replication of picornaviral RNAs were believed to be restricted to the 5' and 3' noncoding regions of the genome. However, an RNA stem-loop in the VP1-coding sequence of human rhinovirus type 14 (HRV-14) is essential for viral minus-strand RNA synthesis (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998). The nucleotide sequence of the apical loop of this internal cis-acting replication element (cre) was critical for RNA synthesis, while secondary RNA structure, but not primary sequence, was shown to be important within the duplex stem. Similar cres have since been identified in other picornaviral genomes. These RNA segments appear to serve as template for the uridylylation of the genome-linked protein, VPg, providing the VPg-pUpU primer required for viral RNA transcription (A. V. Paul et al., J. Virol. 74:10359-10370, 2000). Here, we show that the minimal functional HRV-14 cre resides within a 33-nucleotide (nt) RNA segment that is predicted to form a simple stem-loop with a 14-nt loop sequence. An extensive mutational analysis involving every possible base substitution at each position within the loop segment defined the sequence that is required within this loop for efficient replication of subgenomic HRV-14 replicon RNAs. These results indicate that three consecutive adenosine residues (nt 2367 to 2369) within the 5' half of this loop are critically important for cre function and suggest that a common RNNNAARNNNNNNR loop motif exists among the cre sequences of enteroviruses and rhinoviruses. We found a direct, positive correlation between the capacity of mutated cres to support RNA replication and their ability to function as template in an in vitro VPg uridylylation reaction, suggesting that these functions are intimately linked. These data thus define more precisely the sequence and structural requirements of the HRV-14 cre and provide additional support for a model in which the role of the cre in RNA replication is to act as template for VPg uridylylation.

Base Sequence↗
PubMed2000

Precise mapping of the replication and transcription promoters of human parainfluenza virus type 3.

The terminal RNA regions of the genomic and antigenomic RNAs of the paramyxoviruses and rhabdoviruses are known to contain sequences essential for directing RNA replication and transcription. The 3' terminus (leader region) of the negative-sense, genomic RNA of the rhabdoviruses and paramyxoviruses is known as the leader (Le) promoter and directs synthesis of positive-sense replication and transcription products. The 3' terminus of the antigenome is termed the trailer complementary (TrC) promoter and directs the synthesis of genomic RNA. By creating mutations in the corresponding regions of an HPIV3 minireplicon in which the viral protein coding sequences were replaced by the luciferase gene, we were able to precisely define the elements of the leader promoter involved in directing positive-strand replication of HPIV3. Nucleotides 1 through 12 (from the terminus) formed a domain critical for replication. The region from nucleotides 13 through 55 was important but not crucial for replication, while G residues at positions 79, 85, and 91 comprised another domain critical for replication. It was also shown that the TrC promoter is similar, though not identical, to the Le promoter. Nucleotides 1 through 12 of the TrC promoter were critical for synthesis of genomic RNA, though specific positions behaved differently from the corresponding positions of the Le promoter. While many of these mutations could not be analyzed for transcription because they completely abrogated genomic RNA synthesis (the template for transcription), we were surprised to find that no mutations in the leader promoter which decreased replication had any significant effect on transcription. However, mutations in the intergenic sequence and gene start signal following the leader and preceding the luciferase message severely decreased transcription, but not replication.

Base Sequence↗