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At least 523 records · Page 29Linked to original sources
PubMed1997

Identification of an RNA sequence within an intracisternal-A particle element able to replace Rev-mediated posttranscriptional regulation of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) replication depends on the posttranscriptional regulation by the viral Rev protein and can be replaced with the posttranscriptional RNA control element (CTE) of the type D simian retroviruses. We have identified a sequence which shares only nucleotide sequences of the internal loops and secondary structure with the CTE and which is part of a novel murine intracisternal-A particle (IAP) retroelement, inserted within the transcribed mouse osteocalcin-related gene. This sequence, named CTE(IAP), can replace the Rev-mediated regulation of HIV-1, hence it is a posttranscriptional regulatory element. Related elements have been identified in other IAPs. These results suggest that insertional mutagenesis can affect gene expression by providing a functional posttranscriptional control element. The CTE(IAP) and CTEs of the type D simian retroviruses represent a novel class of RNA elements characterized by unique sequences within the internal loops which are predicted to represent the interaction site with cellular factor(s). These findings suggest that such elements may be involved in posttranscriptional regulation of cellular mRNAs.

Amino Acid Sequence↗
PubMed2004

Forced selection of a human immunodeficiency virus type 1 variant that uses a non-self tRNA primer for reverse transcription: involvement of viral RNA sequences and the reverse transcriptase enzyme.

Human immunodeficiency virus type 1 uses the tRNA(3)(Lys) molecule as a selective primer for reverse transcription. This primer specificity is imposed by sequence complementarity between the tRNA primer and two motifs in the viral RNA genome: the primer-binding site (PBS) and the primer activation signal (PAS). In addition, there may be specific interactions between the tRNA primer and viral proteins, such as the reverse transcriptase (RT) enzyme. We constructed viruses with mutations in the PAS and PBS that were designed to employ the nonself primer tRNA(Pro) or tRNA(1,2)(Lys). These mutants exhibited a severe replication defect, indicating that additional adaptation of the mutant virus is required to accommodate the new tRNA primer. Multiple independent virus evolution experiments were performed to select for fast-replicating variants. Reversion to the wild-type PBS-lys3 sequence was the most frequent escape route. However, we identified one culture in which the virus gained replication capacity without reversion of the PBS. This revertant virus eventually optimized the PAS motif for interaction with the nonself primer. Interestingly, earlier evolution samples revealed a single amino acid change of an otherwise well-conserved residue in the RNase H domain of the RT enzyme, implicating this domain in selective primer usage. We demonstrate that both the PAS and RT mutations improve the replication capacity of the tRNA(1,2)(Lys)-using virus.

Amino Acid Substitution↗
PubMed2002

Direct measurement of the association constant of HER2/neu antisense oligonucleotide to its target RNA sequence using a molecular beacon.

A molecular beacon approach was developed to directly determine the association constant of RNA-DNA hybrid formation. The molecular beacon was composed of a 15-nt loop structure containing the antisense sequence that can hybridize with the AUG translational start site of the HER2/neu gene, which is overexpressed in a significant proportion of breast, ovarian, and lung tumors. The equilibrium association constant (Ka) of DNA binding to the RNA oligonucleotide was 6.4 +/- 0.14 x 10(7) M(-1) in the presence of 150 mM NaCl at 22 degrees C. The free energy change (AG) associated with RNA-DNA hybrid formation was -10.7 kcal/mole. The melting temperature (Tm) of RNA-DNA hybrid was 64.4 degrees C +/- 1 degree C in the presence of 150 mM NaCl. The RNA-DNA hybrid was more stable than the corresponding DNA-DNA duplex in 150 mM NaCl, as judged by both Ka and Tm data. We also determined the Ka, deltaG, and Tm values of RNA-DNA and DNA-DNA duplex formation in the presence of three monovalent cations, Li+, K+, and Cs+. The feasibility of this method was also investigated using a phosphorothioate molecular beacon. The information generated through this new approach for thermodynamic measurements might be useful for the design of oligonucleotides for antisense therapeutics.

Cesium↗
PubMed1976

Adenovirus type 12-specific RNA sequences during productive infection of KB cells.

The complementary strands of adenovirus type 12 DNA were separated, and virus-specific RNA was analyzed by saturation hybridization in solution. Late during infection whole cell RNA hybridized to 75% of the light (1) strand and 15% of the heavy (H) strand, whereas cytoplasmic RNA hybridized to 65% of the 1 strand and 15% of the h strand. Late nuclear RNA hybridized to about 90% of the 1 strand and at least 36% of the h strand. Double-stranded RNA was isolated from infected cells late after infection, which annealed to greater than 30% of each of the two complementary DNA strands. Early whole cell RNA hybridized to 45 to 50% of the 1 strand and 15% of the h strand, whereas early cytoplasmic RNA hybridized to about 15% of each of the complementary strands. All early cytoplasmic sequences were present in the cytoplasm at late times.

Adenoviridae↗
PubMed1995

Saponin pre-treatment in pre-embedding electron microscopic in situ hybridization for detection of specific RNA sequences in cultured cells: a methodological study.

We describe a method for detection of specific RNA targets in cultured cells at the electron microscopic (EM) level using pre-embedding in situ hybridization (ISH). The specimens were monitored by reflection-contrast microscopy (RCM) before processing for EM. A good balance between preservation of ultrastructure and intensity of hybridization signals was obtained by using mild aldehyde fixation followed by saponin permeabilization. Digoxigenin-labeled probes were used for detection of human elongation factor (HEF) mRNA in HeLa cells, immediate early (IE) mRNA in rat 9G cells, and 28S rRNA in both cell lines. The hybrids were detected immunocytochemically by the peroxidase/diaminobenzidine (DAB) method or by ultra-small gold with silver enhancement. Comparison of these methods favored the peroxidase/DAB system. The accessibility of RNA in the different cell compartments was dependent on the extent of cross-linking during primary fixation even after permeabilization with saponin. By using the most optimal ISH protocol and the peroxidase/DAB system, we detected 28S rRNA over all ribosomes in the cytoplasm but not in the nucleoli, and IE mRNA in a large spot with many smaller spots around it in the nucleoplasm as well as in speckles over the cytoplasm. The sensitivity of the method is such that HEF housekeeping gene transcripts were detected in the cytoplasm.

Animals↗
PubMed2005

Genomic RNA sequence of Feline coronavirus strain FIPV WSU-79/1146.

A consensus sequence of the Feline coronavirus (FCoV) (strain FIPV WSU-79/1146) genome was determined from overlapping cDNA fragments produced by RT-PCR amplification of viral RNA. The genome was found to be 29 125 nt in length, excluding the poly(A) tail. Analysis of the sequence identified conserved open reading frames and revealed an overall genome organization similar to that of other coronaviruses. The genomic RNA was analysed for putative cis-acting elements and the pattern of subgenomic mRNA synthesis was analysed by Northern blotting. Comparative sequence analysis of the predicted FCoV proteins identified 16 replicase proteins (nsp1-nsp16) and four structural proteins (spike, membrane, envelope and nucleocapsid). Two mRNAs encoding putative accessory proteins were also detected. Phylogenetic analyses confirmed that FIPV WSU-79/1146 belongs to the coronavirus subgroup G1-1. These results confirm and extend previous findings from partial sequence analysis of FCoV genomes.

Coronavirus, Feline↗
PubMedDate not supplied

[Absence of echoviral RNA sequences in medulla oblongata samples taken from patients who died of ALS].

BACKGROUND AND PURPOSE: The role of viral infection in the pathogenesis of ALS has been raised many times in the previous papers. The presence of an enterovirus genome has previously been confirmed using PCR RT and PCR in situ in spinal cord tissue samples taken from patients who died of ALS. Viral genome sequencing has also been used to show 91% and 88% homogeneity with ECHO 6 and 7 viruses, respectively. A year later the same method did not confirm the presence of the virus in spinal cord fragments taken from 30 patients who died of ALS nor in an 18 person control group. The present study was aimed to find persistent ECHO 6 and 7 viral infections in tissue samples taken from patients who died of ALS. MATERIAL AND METHODS: RNA was isolated from frozen medulla oblongata samples taken from six patients who died of ALS (hospitalized in the Neurological Clinic CSK AM in the years 2000-2002). The presence of RNA was confirmed using RNA beta-actine. Oligo 2 and 3 as well as pEforward and pErevers primers were used for amplification. RESULTS: All samples returned negative results. CONCLUSIONS: In the samples studied no correlation was found between ECHO 6 and 7 viral infections and ALS.

Actins↗
PubMed1983

"Silent" nucleotide substitution in codon 24 of a beta+ thalassemia globin gene activates splice site in coding sequence RNA.

A beta+-thalassemia globin gene was isolated from the genome of a black patient by molecular cloning. DNA sequence analysis revealed only a single difference between this gene and the normal human beta-globin gene--adenine is substituted for thymine in the third position of codon 24. This mutation is silent at the protein sequence level. We compared the function of this beta+-thalassemia gene with the normal human beta-globin gene in monkey kidney cells using plasmid expression vectors. The codon 24 substitution activates a 5' splice site that involves the guanine-thymine dinucleotide present in codon 25, 16 nucleotides upstream from the normal exon 1-intron I boundary. Splices at the abnormal 5' site in the coding sequence are completed with the normal 3' splice site at the end of intron I. This splicing abnormality leads to a fourfold decrease in the accumulation of normally processed beta-globin mRNA, thereby causing the beta+-thalassemia phenotype.

Base Sequence↗
PubMed1974

Sarcoma-virus-related RNA sequences in normal rat cells.

A rat type C virus spontaneously activated from the NRK (normal rat kidney) cell line was found to have two major size classes of viral RNA subunits sedimenting at 35 and 30 S. Virus-producing cells contained both RNA species, while normal "virus-free" rat cells contained primarily virus-specific 30S RNA species. A DNA transcript, specific for Kirsten sarcoma virus, prepared from virus activated in nonproducer BALB/c cells originally transformed by Kirsten sarcoma virus and rendered specific for the virus by absorption of sequences related to mouse helper virus hybridized only with the 30S RNA species of virus-producing rat cells and normal rat cells. These findings are consistent with the hypothesis that sarcoma-specific nucleic acid sequences in kirsten sarcoma virus emerged through a process that incorporated some portions of 30S RNA species from rat cells (either normal or virus-producing) into the Kirsten leukemia virus during passage in vivo of that virus. The virus designated M-MSV(RaLV), which originally derived from tumor induced by Moloney sarcoma virus (M-MSV) in rats, contained 35S RNA species of rat type C viruses and 30S RNA species specific for both rat and mouse viruses. It appears striking that for these two animal species, sarcoma-virus-specific information resides on a 30S subunit.

Animals↗
PubMed1986

Detection of Coxsackie-B-virus-specific RNA sequences in myocardial biopsy samples from patients with myocarditis and dilated cardiomyopathy.

Full-length virus genomic RNA (7.4 kilobases; kb) was isolated from Coxsackie B2 virus purified from infected monkey kidney cells in culture. DNA complementary to 6.3 kb of virus RNA was prepared by reverse transcription and cloned in a plasmid vector. A 1.6 kb Coxsackie-B-virus-specific DNA clone derived from the conserved 3' region of the virus genome was used as a hybridisation probe to test for the presence of virus nucleic acid sequences in myocardial biopsy samples. Positive hybridisation signals, quantified by densitometry, were obtained with 9 of 17 samples from patients with histological evidence of active or healing myocarditis or dilated cardiomyopathy with inflammatory changes. No Coxsackie-B-virus-specific sequences were detected in 4 samples from patients in whom a viral aetiology was unlikely and the histological diagnosis was negative for myocarditis.

Adolescent↗
PubMed2006

Identification of c-myc coding region determinant RNA sequences and structures cleaved by an RNase1-like endoribonuclease.

The coding region of c-myc mRNA encompassing the coding region determinant (CRD) nucleotides (nts) 1705-1792 is critical in regulating c-myc mRNA stability. This is in part due to the susceptibility of c-myc CRD RNA to attack by an endoribonuclease. We have previously purified and characterized a mammalian endoribonuclease that cleaves c-myc CRD RNA in vitro. This enzyme is tentatively identified as a 35 kDa RNase1-like endonuclease. In an effort to understand the sequence and secondary structure requirements for RNA cleavage by this enzyme, we have determined the secondary structure of the c-myc CRD RNA nts 1705-1792 using RNase probing technique. The secondary structure of c-myc CRD RNA possesses five stems; two of which contain 4 base pairs (stems I and V) and three consisting of 3 base pairs (stems II, III, and IV). Endonucleolytic assays using the c-myc CRD and several c-myc CRD mutants as substrates led to the following conclusions: (i) the enzyme prefers to cleave in between the dinucleotides UA, CA, and UG in single-stranded regions; (ii) the enzyme is more specific towards UA dinucleotides. These properties further distinguish the enzyme from previously described mammalian endonuclease that cleaves c-myc mRNA in vitro.

Base Sequence↗
PubMed1983

"Silent" nucleotide substitution in a beta+-thalassemia globin gene activates splice site in coding sequence RNA.

A beta+-thalassemia globin gene was isolated from the genome of a Black individual by molecular cloning. DNA sequence analysis revealed only a single difference between this gene and the normal human beta-globin gene--adenine is substituted for thymine in the third position of codon 24. Codon 24 in both the normal gene (GGT) and the beta+-thalassemia gene (GGA) encodes glycine. The function of this beta+-thalassemia gene was compared to the function of the normal human beta-globin gene in monkey kidney cells by using plasmid expression vectors. The codon 24 substitution activates a 5' splice site that involves the guanine-thymine dinucleotide present in codon 25, 16 nucleotides upstream from the normal exon 1-intron I boundary. The splice, involving the abnormal 5' site in codon 25, is completed with the normal 3' splice site at the end of intron I. This splicing abnormality leads to a 75% decrease in the accumulation of normally processed beta-globin mRNA, thereby causing the beta+-thalassemia phenotype.

Base Sequence↗
PubMed1997

Ribosomal RNA sequencing reveals differences between the genotypes of Giardia isolates recovered from humans and dogs living in the same locality.

A polymerase chain reaction-based method for genotyping Giardia duodenalis isolates using a polymorphic region near the 5' end of the small subunit ribosomal (SSU) RNA gene is described. Analysis was performed using Giardia cysts purified directly from feces. Isolates were collected from humans and dogs living in isolated Aboriginal communities where Giardia infections are highly endemic. This is the first report of the genetic characterization of Giardia from dogs and humans living in the same locality. Comparison of the SSU-rRNA sequences from 13 human and 9 dog isolates revealed 4 different genetic groups. Groups 1 and 2 contained all of the human isolates, whereas groups 3 and 4 consisted entirely of Giardia samples recovered from dogs. One dog sample contained templates from both groups 2 and 3. These results suggest that zoonotic transmission of Giardia infections between humans and dogs does not occur frequently in these communities. The dog-associated SSU-rRNA sequences have not been reported before, suggesting a new G. duodenalis subgroup. A genetic basis for the differences observed between the groups was supported by sequence analysis of 9 in vitro cultured isolates that were placed into the same genetic groups established by enzyme electrophoresis.

Animals↗
PubMed2001

Gyrodactyloides bychowskii (Monogenea: Gyrodactylidae) from sea-caged Atlantic salmon Salmo salar in Scotland: occurrence and ribosomal RNA sequence analysis.

Gyrodactyloides bychowskii has been recorded in Scottish waters for the first time. The parasite was found on the gills of Atlantic salmon reared in seawater. An integrated morphological and molecular examination of the parasite was carried out. Prevalence of the parasite was greatest in February and declined to 0 by June in 1999. During 2000, parasites were located in March and November. An overdispersion of parasites was recorded, with intensity of infection reaching over 200 parasites per gill arch in some fish. Parasitised gill tissue showed hyperplasia and hypertrophy but not always at the site of parasite attachment. The internal transcribed spacer of the ribosomal RNA gene array was amplified by PCR and sequenced. This sequence shared greatest similarity with the internal transcribed spacer of Gyrdicotylus gallieni, followed by Gyrodactylus species. This is the first molecular analysis of this parasite and provides sequence data that may be used in comparison of G. bychowskii from other locations or in phylogenetic analysis of this group of Monogenea.

Animals↗
PubMed1999

A potential mechanism for selective control of cap-independent translation by a viral RNA sequence in cis and in trans.

Highly efficient cap-independent translation initiation at the 5'-proximal AUG is facilitated by the 3' translation enhancer sequence (3'TE) located near the 3' end of barley yellow dwarf virus (BYDV) genomic RNA. The role of the 3'TE in regulating viral translation was examined. The 3'TE is required for translation and thus replication of the genomic RNA that lacks a 5' cap (Allen et al., 1999, Virology253:139-144). Here we show that the 3'TE also mediates translation of uncapped viral subgenomic mRNAs (sgRNA1 and sgRNA2). A 109-nt viral sequence is sufficient for 3'TE activity in vitro, but additional viral sequence is necessary for cap-independent translation in vivo. The 5' extremity of the sequence required in the 3' untranslated region (UTR) for cap-independent translation in vivo coincides with the 5' end of sgRNA2. Thus, sgRNA2 has the 3'TE in its 5' UTR. Competition studies using physiological ratios of viral RNAs showed that, in trans, the 109-nt 3'TE alone, or in the context of 869-nt sgRNA2, inhibited translation of genomic RNA much more than it inhibited translation of sgRNA1. The divergent 5' UTRs of genomic RNA and sgRNA1 contribute to this differential susceptibility to inhibition. We propose that sgRNA2 serves as a novel regulatory RNA to carry out the switch from early to late gene expression. Thus, this new mechanism for temporal control of translation control involves a sequence that stimulates translation in cis and acts in trans to selectively inhibit translation of viral mRNA.

3' Untranslated Regions↗
PubMed2002

A pregenomic RNA sequence adjacent to DR1 and complementary to epsilon influences hepatitis B virus replication efficiency.

Hepatitis B virus (HBV) possesses a 3.2-kb partially double-stranded DNA genome that is generated inside the nucleocapsid by the reverse transcription of the 3.5-kb pregenomic viral transcript. The initial steps in viral replication involve the recognition of an encapsidation signal termed epsilon (epsilon) at the 5'-end of the pregenomic RNA by the HBV polymerase. The polymerase-bound pregenomic RNA is subsequently incorporated into an immature nucleocapsid particle and minus-strand HBV DNA synthesis is initiated utilizing the bulge region of epsilon as a template and a tyrosine residue in the amino-terminal region of the polymerase as a primer. Three nucleotides complementary to the 3'-end of the bulge region of epsilon are synthesized and subsequently translocated with the polymerase molecule to the acceptor site located in the DR1 sequence present at the 3'-end of the pregenomic RNA. Using mutagenesis analysis, a sequence element designated phi (phi) located upstream of the 3' DR1 sequence has been identified that is complementary to epsilon and is important for efficient viral replication. This element may bring the 3' DR1 sequence into proximity with the three nucleotide primer synthesized at the bulge of epsilon and facilitate primer translocation to the 3' DR1 acceptor sequence. Sequence elements with similar proximity to the 3' DR1 sequences and complementarity to epsilon are present in the woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV), suggesting the phi regulatory element may be phylogenetically conserved due to its functional importance in hepadnavirus minus-strand DNA synthesis.

3' Untranslated Regions↗
PubMed1995

Interactions between the IBV nucleocapsid protein and RNA sequences specific for the 3' end of the genome.

The infectious bronchitis virus (IBV) nucleocapsid protein was expressed as a fusion protein in bacteria. The coding sequence differed from the native protein only in the addition of six histidine residues at the amino terminus which were used for enrichment with a nickel affinity column. In gel shift assays, the mobility of labelled G RNA was decreased with increasing concentrations of the fusion protein. Competitive gel shift assays with labelled G RNA indicated that the protein interacted with relatively high avidities to several unlabelled RNAs representing sequences at the 3' noncoding end of the IBV genome. Cache Valley virus (a bunyavirus) mRNA transcribed from the smaller segment cDNA also inhibited the interaction with IBV G RNA to the same extent as homologous unlabelled G RNA. In contrast, interactions of the fusion proteins with a region from 99 to 249 bases from the 3' terminus of the IBV genome and bovine liver RNA were relatively weak. The binding of IBV nucleocapsid protein with RNA probably requires specific sequences and/or structures that are present at a number of sites on the genome, and may represent a common mechanism used by similar viral proteins whose functions depend on binding to RNA.

Animals↗