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At least 343 records · Page 19Linked to original sources

PubMed1975

Strand-selective transcription of globin genes in rabbit erythroid cells and chromatin.

In order to investigate the symmetry of globin gene transcription, complementary RNA (cRNA) was synthesized using rabbit globin complementary DNA (cDNA) as a template for Escherichia coli DNA-dependent RNA polymerase (RNA nucleotidyltransferase). The cRNA hybridized specifically to its own cDNA template but not to sheep cDNA, rabbit globin mRNA, or poly(dT). Hybridization studies with cRNA demonstrated that RNA sequences transcribed from the DNA strand complementary to the globin gene region (anti-strand) were not present in cellular, total nuclear, or fractionated nuclear RNA from rabbit marrow. Such sequences were detected in RNA transcribed from rabbit marrow chromatin by E. coli or sheep liver RNA polymerases, but amounted to less than 50% of the globin mRNA sequences present in the same transcript. The evidence indicates that globin mRNA transcription is predominantly DNA strand specific.

Animals↗

PubMed1975

Rous sarcoma virus activates embryonic globin genes in chicken fibroblasts.

Complementary DNA (cDNA) specific for chick globin mRNA sequences fails to hybridize to total RNA extracted from chicken fibroblasts. After infection by Rous sarcoma virus, RNA complementary to globin cDNA is detectable in 100-500 copies per cell. Infection of fibroblasts with the transformation defective (td) deletion mutant of Rous sarcoma virus leads to normal virus production, but not to host cell transformation or accumulation of RNA sequences complementary to globin cDNA. Our evidence shows that the globin genes activated by Rous sarcoma virus are those specified by embryonic chick red cells; adult-specific globin sequences were not detected.

Age Factors↗

PubMed1986

Inhibition of replication and expression of human T-cell lymphotropic virus type III in cultured cells by exogenous synthetic oligonucleotides complementary to viral RNA.

The possibility of using oligodeoxynucleotides complementary to viral RNA or proviral DNA to inhibit the replication of human T-cell lymphotropic virus type III (HTLV-III) [the etiological agent of acquired immunodeficiency syndrome (AIDS)] in cultured human cells was addressed by studying the association of 32P-labeled oligodeoxynucleotides with mammalian cellular components. The results indicated that exogenous oligodeoxynucleotides at 20 microM became associated with the membrane/cytosol fractions of the cell in amounts approximating 1.5 microM. Oligodeoxynucleotides complementary to a region close to the tRNALys primer binding site on HTLV-III RNA and others complementary to HTLV-III mRNA donor or acceptor splice sites inhibited viral replication (assayed as reverse transcriptase) and gene expression (assayed as virus-encoded proteins p15 and p24) by as much as 95%. Use of control (random) oligodeoxynucleotides suggests that the antiviral effects were specific. Although these results pertain to HTLV-III-infected cells in tissue culture, rather than to AIDS patients, they nevertheless point to a therapeutic potential of the complementary oligodeoxynucleotide ("hybridization competition" or "hybridon") approach in the treatment of patients with AIDS and AIDS-related complex.

Base Sequence↗

PubMed1979

Characterization of virus-like particles produced by an influenza A virus.

The influenza strain 413 1,1 segregated as a stable recombinant during passage of the isolate 19/N which was obtained after double infection of chick embryo fibroblasts by virus N and the fowl plague virus (FPV) mutant ts 19. Its gene constellation was determined by molecular hybridization. Upon infection of chick embryo cells by this recombinant strain, two particle populations of high (H) and low (L) buoyant densities were produced. By biological and biochemical parameters, the H-population (delta = 1.22 g/cm3) cannot be distinguished from standard infectious influenza virus. In contrast, the noninfectious L-particles (delta = 1.14 g/cm3) lack all virus-specific glycoproteins (HA, NA) as well as the matrix protein M and are visualized by electron microscopy as spikeless particles. Significant changes in the quantitative composition of the phospholipid bilayer are evident as compared to the H-particles. In addition to the previously characterized eight genes both populations contain a variety of smaller RNA fragments which hybridize with complementary RNA and presumably represent degradation products of full-length genes.

Animals↗

PubMed2005

Oligonucleotide-microarray analysis of peripheral-blood lymphocytes in severe asthma.

CD4 + lymphocytes play a key role in asthma pathogenesis, but much remains unknown about the genetic mechanisms that affect disease severity. In this study we sought to investigate global patterns of gene expression in CD4 + lymphocytes isolated from subjects with severe asthma through the use of microarray technology. CD4 + lymphocytes were separated from peripheral blood, total RNA was purified, and biotinylated complementary RNA was prepared and hybridized to Affymetrix HU133 chips (Affymetrix, Santa Clara, Calif). Using the robust multi-chip average procedure, we compared the messenger RNA expression profiles of more than 33,000 genes of CD4 + lymphocytes in subjects with severe ( n = 5) and mild ( n = 5) asthma. Forty genes had 2-fold mean expression differences or greater. Thirty-seven genes were up-regulated, including transforming growth factor-beta and those involved in T-cell activation, proliferation, and cytoskeletal changes. Three genes were down-regulated, including the T-cell-receptor delta locus. This study demonstrates a method by which CD4 + lymphocytes can be extracted from blood for the purpose of microarray analysis. Furthermore, we show that T-lymphocytes from the peripheral blood of subjects with severe and mild asthma differ in their gene-expression profiles, supporting the view that asthma is a systemic disease. These differentially expressed genes identify potential molecular targets for preventive and therapeutic options for severe asthma.

Adult↗

PubMed1987

In vitro synthesis of influenza viral RNA: characterization of an isolated nuclear system that supports transcription of influenza viral RNA.

An in vitro system for the synthesis of influenza viral RNA was developed using isolated nuclei prepared from influenza virus-infected HeLa cells. In this system, two species of positive-sense RNA, i.e., mRNA and cRNA (complementary RNA to vRNA), were found to be synthesized when analyzed by RNA-RNA hybridization using a minus-strand RNA probe and high resolution gel electrophoresis. The in vitro RNA synthesis required Mg2+, GTP, CTP, UTP, a high concentration of ATP, and an ATP regenerating system. Neither actinomycin D nor alpha-amanitin, potent inhibitors for cellular DNA-dependent RNA polymerases, inhibited the RNA synthesis. Addition of ApG or capped RNA, well-known primers for virion-associated RNA polymerase, markedly enhanced the extent of RNA synthesis. The maximum activity was observed with nuclei isolated from cells at 5 h after infection. This system is useful for the purification and characterization of factors involved in the transcription of these two species positive-sense RNA.

Adenosine Triphosphate↗

PubMed1980

Interferon induction by viruses. IV. Sindbis virus: early passage defective-interfering particles induce interferon.

We have shown that a single defective-interfering (DI) particle of early (5th) passage Sindbis virus induces maximal amounts of interferon in an 'aged' primary chick embryo cell. The capacity of such DI particles to induce interferon is inactivated by small amounts of u.v. radiation (i/e dose = 232 ergs/mm2). The I/e dose for inactivation of the interferon-inducing capacity of infectious virus particles is 399 ergs/mm2 and for infectivity is 101 ergs/mm2. Pre-treatment with interferon blocks formation of interferon in response to either DI or infectious virus particles. Our results suggest that Sindbis virus genes must be expressed to form the interferon inducer, which is presumably a molecule of double-stranded (ds)RNA. We postulate that for interferon induction, the genomic RNA which codes for genes G and A must be translated into products whose concerted action produces a dsRNA molecule upon synthesis of a segment of RNA complementary to the genome. The RNA from early passage DI particles is sufficiently large (25S, 1.6 x 10(6) mol. wt.) to accommodate these genes, whereas the RNA from the late passage DI particles (20S, 1.0 x 10(6) mol. wt.) is not. Late (15th) passage DI particles do not induce interferon formation.

Animals↗

PubMed1980

Short-lived minus-strand polymerase for Semliki Forest virus.

Semliki Forest virus (SFV)-infected BHK-21, Vero, and HeLa cells incorporated [3H]uridine into 42S and 26S plus-strand RNA and into viral minus-strand RNA (complementary to the 42S virion RNA) early in the infectious cycle. Between 3 and 4 h postinfection, the synthesis of minus-strand RNA ceased in these cultures, although the synthesis of plus-strand RNA continued at a maximal rate. At the time of cessation of minus-strand RNA synthesis, two changes in the pattern of viral protein synthesis were detected: a decrease in the translation of nonstructural proteins and an increase in the translation of the viral structural proteins. Addition of cycloheximide and puromycin to cultures of SFV-infected BHK cells actively synthesizing both viral plus- and minus-strand RNA resulted within 15 to 30 min in the selective shutoff of minus-strand RNA synthesis. Removal of the cycloheximide-containing medium led to the resumption of minus-strand synthesis and to an increased rate of viral RNA synthesis. We conclude that the minus-strand polymerase regulates the rate of SFV plus-strand RNA synthesis by determining the number of minus-strand templates and that the synthesis of the minus-strand templates is regulated at the level of translation by a mechanism which utilizes one or more short-lived polymerase proteins.

Animals↗

PubMed1996

Saccharomyces cerevisiae L-BC double-stranded RNA virus replicase recognizes the L-A positive-strand RNA 3' end.

L-A and L-BC are two double-stranded RNA viruses present in almost all strains of Saccharomyces cerevisiae. L-A, the major species, has been extensively characterized with in vitro systems established, but little is known about L-BC. Here we report in vitro template-dependent transcription, replication, and RNA recognition activities of L-BC. The L-BC replicase activity converts positive, single-stranded RNA to double-stranded RNA by synthesis of the complementary RNA strand. Although L-A and L-BC do not interact in vivo, in vitro L-BC virions can replicate the positive, single-stranded RNA of L-A and its satellite, M1, with the same 3' end sequence and stem-loop requirements shown by L-A virions for its own template. However, the L-BC virions do not recognize the internal replication enhancer of the L-A positive strand. In a direct comparison of L-A and L-BC virions, each preferentially recognizes its own RNA for binding, replication, and transcription. These results suggest a close evolutionary relation of these two viruses, consistent with their RNA-dependent RNA polymerase sequence similarities.

Base Sequence↗

PubMed2000

Activation of the interferon-inducible 2'-5'-oligoadenylate synthetase gene by hepatitis C virus core protein.

The effects of hepatitis C virus (HCV) proteins on several signal transduction pathways in human nonneoplastic hepatocyte PH5CH8 cells were investigated using expression vectors encoding HCV proteins derived from HCV-infected human nonneoplastic cultured T-lymphocyte and hepatocyte cells (MT-2C and PH5CH7), which could support HCV replication. The amino acid sequences of HCV proteins obtained from HCV-infected human cells were identical or very close to the consensus sequences of the proteins derived from the original inoculum used for HCV infection. During the course of the study, we found that HCV core protein specifically activated the 40/46-kDa 2'-5'-oligoadenylate synthetase (2'-5'-OAS) gene promoter in a dose-dependent manner in different human hepatocyte cell lines (PH5CH8, HepG2, and PLC/PRF/5). We also found that the activation by core protein was further enhanced in the cells treated with alpha interferon. The expression of E1 or E2 envelope protein or nonstructural NS5A protein did not activate the 2'-5'-OAS gene promoter. We demonstrated that the activation by core protein in the hepatocyte cells was suppressed by antisense RNA complementary to core-encoding RNA. Deletion mutant analysis of core protein and deletion analysis of the 2'-5'-OAS gene promoter have been performed. Finally, we demonstrated that the activation of the 2'-5'-OAS gene occurred at the transcriptional level and furthermore demonstrated that the endogenous 2'-5'-OAS gene was also activated by core protein. This is the first report to show that a viral protein activated the 2'-5'-OAS gene.

2',5'-Oligoadenylate Synthetase↗

PubMed2003

Reverse genetics for crimean-congo hemorrhagic fever virus.

The widespread geographical distribution of Crimean-Congo hemorrhagic fever (CCHF) virus (more than 30 countries) and its ability to produce severe human disease with high mortality rates (up to 60%) make CCHF a major public health concern worldwide. We describe here the successful establishment of a reverse genetics technology for CCHF virus, a member of the genus Nairovirus, family BUNYAVIRIDAE: The RNA polymerase I (pol I) system was used to generate artificial viral RNA genome segments (minigenomes), which contained different reporter genes in antisense (virus RNA) or sense (virus-complementary RNA) orientation flanked by the noncoding regions of the CCHF virus S segment. Reporter gene expression was observed in different eukaryotic cell lines following transfection and subsequent superinfection with CCHF virus, confirming encapsidation, transcription, and replication of the pol I-derived minigenomes. The successful transfer of reporter gene activity to fresh cells demonstrated the generation of recombinant CCHF viruses, thereby confirming the packaging of the pol I-derived minigenomes into progeny viruses. The system offers a unique opportunity to study the biology of nairoviruses and to develop therapeutic and prophylactic measures against CCHF infections. In addition, we demonstrated for the first time that the human pol I system can be used to develop reverse genetics approaches for viruses in the family BUNYAVIRIDAE: This is important since it might facilitate the manipulation of bunyaviruses with cell and host tropisms restricted to primates.

Animals↗

PubMed1985

Dysdifferentiative nature of aging: passage number dependency of globin gene expression in normal human diploid cells grown in tissue culture.

Aging may be a result of cells drifting away from their proper state of differentiation. This process has been called dysdifferentiation. Normal diploid cells grown in tissue culture conditions undergo numerous biochemical and morphological changes and have a finite division potential. These changes could be a result of such a dysdifferentiation process. Changes in the differentiated state of a cell are frequently manifested by the expression of genes that are normally repressed. Previous studies have shown about a two-fold age-dependent increase of alpha and beta globin-like RNA in mouse brain and liver tissues. Therefore, the possible presence and increase of globin RNA was investigated in the nonerythroid human diploid strain WI-38 grown in tissue culture as a function of population doublings. A DNA X RNA hybridization technique using specific complementary DNA (cDNA) to alpha and beta human globin was used to detect possible complementary RNA sequences in total cellular RNA preparations extracted from cells at population doublings of 26.4 and 46. No globin-like RNA sequences could be detected above background noise levels for either of these two passage numbers. Thus, the globin RNA genes appear to be highly repressed and this degree of repression maintained as the culture approaches its characteristic population doubling limit.

Aging↗

PubMed2004

Genetically targeted cancer therapy: tumor destruction by PKR activation.

The is a double-stranded RNA-activated protein kinase (PKR) has been largely investigated for its key role in viral host defense. Although best characterized by its function in mediating the antiviral and antiproliferative effects of interferon (IFN), PKR is also implicated in transcriptional regulation, cell differentiation, signal transduction, and tumor suppression. However, recent findings identifying PKR as an important effector of apoptosis have led to an increased interest in PKR modulation as an antitumor strategy. PKR can either be up-regulated through direct induction by the transcription factor E2F-1, or it can be activated through direct protein-protein interactions with the melanoma differentiation-associated gene-7 (MDA7, IL-24). Additionally, the intracellular formation of double-stranded RNA by transfection with antisense RNA complementary to tumor-specific RNA sequences can induce PKR activation and apoptosis selective to these tumor cells. The growing application of viral vector-based gene therapies and oncolytic, replicating viruses that must elude viral defense in order to be effective, has also drawn attention to PKR. Oncolytic viruses, like the attenuated herpes simplex virus R3616, the vesicular stomatitis virus, or reovirus, specifically replicate in tumor cells only because the viral host defense in the permissive cells is suppressed. In this article we review the role of PKR as an effector of apoptosis and a target for tumor treatment strategies and discuss the potential of PKR-modifying agents to treat patients with cancer. Targeted gene therapy against cancer can be approached by activation of PKR with the down-regulation of protein synthesis and induction of apoptosis, or by suppression of PKR with the propagation of oncolytic virus. Since the PKR pathway can be modified by many routes, antitumor therapies combining oncolytic virus, gene therapies, and chemotherapy with PKR modifiers are likely to emerge in the near future as therapeutic options in the treatment of patients with cancer.

Animals↗

PubMedDate not supplied

Analysis of the genome structure of influenza A virus strains isolated during the epidemics of 1977-1980.

The genome structure of different A(H1N1) and A(H3N2) influenza virus strains isolated from 1977 to 1980 was analysed by electrophoresis of single-stranded RNA and of the double-stranded RNA obtained by hybridization with complementary RNA of the A/Stavropol 188/79 (H3N2) strain. The differences in genome structure are insignificant within the A(H1N1) subtype, and considerable (involving almost all the 8 RNA fragments) between the two A(H3N2) strains (A/Texas 1/77 and A/Stavropol 188/79). The great differences between the genomes of A(H1N1) and A(H3N2) strains do not exclude certain genetic relationships between these viruses.

Base Sequence↗

PubMed1986

GABAergic regulation of enkephalin in rat striatum: alterations in Met5-enkephalin level, precursor content and preproenkephalin messenger RNA abundance.

The influence of chronic activation of gamma-aminobutyric acid (GABA) system on Met5-enkephalin (ME) biosynthesis was investigated in male rats. Activation of GABA system was achieved by raising the brain GABA concentration with aminooxyacetic acid (AOAA) or gabaculine which inhibits GABA-transaminase the enzyme responsible for the catabolism of GABA. After a regimen of repeated administration of AOAA (80 mg/kg/day for 8 days), the GABA concentration in the striatum could be maintained 2-fold greater than control value. AOAA decreased the ME levels in the striatum in a dose (20, 40 and 80 mg/kg)- and time (1-, 2-, 4- and 8-day treatment(s)-dependent fashion. Gabaculine also decreased the ME level in the striatum. Changes in ME level were not observed in other brain regions such as hypothalamus, hippocampus, frontal cortex and medulla/pons. In order to understand the mechanism involved in the decrease in ME level, the biosynthesis of ME was assessed. The preproenkephalin messenger RNA abundance was quantitated by RNA-complementary DNA hybridization technique; the total RNA was isolated, dot-blotted and hybridized with a nick-translated complementary DNA probe from rat brain. Administration of AOAA (80 mg/kg/day) for 8 days increased the preproenkephalin messenger RNA abundance whereas 1-, 2- or 4-day treatments did not alter the levels significantly. A similar trend of response was observed in the cryptic ME level which is indicative of the precursor content. The results suggest that chronic activation of the GABA system induces a sustained release of ME; in order to replenish the depletion, the biosynthesis of ME is augmented.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminooxyacetic Acid↗

PubMed2000

Synthesis and hybridization properties of polyamide based nucleic acid analogues incorporating pyrrolidine-derived nucleoamino acids.

Ndelta-Fmoc protected nucleoamino acids of type I (Base = T, C, A) have been synthesized and employed as building blocks for the construction of novel polyamide based nucleic acid analogues. Homopyrimidine oligomer A binds to complementary RNA with significant affinity and in a sequence-specific fashion, while no binding was observed to complementary DNA.

Amino Acids↗

PubMed2002

Synthesis and hybridization properties of RNA containing 8-chloroadenosine.

8-Chloroadenosine (8-Cl-Ado) has shown potential as a chemotherapeutic agent for the treatment of multiple myeloma and certain leukemias. 8-Cl-Ado treatment leads to a decrease in global RNA levels and incorporation of the analog into cellular RNA in malignant cells. To investigate the effects of 8-Cl-Ado modifications on RNA structure and function, an 8-Cl-Ado phosphoramidite and controlled-pore glass support were synthesized and used to introduce 8-Cl-Ado at internal and 3'- terminal positions, respectively. RNA oligonucleotides containing 8-chloroadenine (8-Cl-A) residues were synthesized and hybridized with complementary RNA strands. Circular dichroism spectroscopy of the resulting RNA duplexes revealed that the modified nucleobase does not perturb the overall A-form helix geometry. The thermal stabilities of 8-Cl-Ado modified duplexes were determined by UV thermal denaturation analysis and were compared with analogous natural duplexes containing standard and mismatched base pairs. The 8-Cl-Ado modification destabilizes RNA duplexes by approximately 5 kcal/mole, approximately as much as a U:U mismatched base pair. The duplex destabilization of 8-Cl-A may result from perturbation of Watson-Crick base pairing induced by conformational preferences of 8-halogenated nucleosides.

2-Chloroadenosine↗

PubMed1992

Inhibition of mouse hepatitis virus multiplication by an oligonucleotide complementary to the leader RNA.

An oligonucleotide complementary to a leader RNA of positive-stranded mouse hepatitis virus (MHV) was tested for the effect on the viral multiplication in mouse DBT cells. A 14-mer antisense oligonucleotide contained a sequence complementary to the conserved pentanucleotide sequence, UCUAA, of the leader RNA. A treatment of MHV-infected cells with the antisense oligonucleotide at concentrations from 5 to 25 microM had an inhibitory effect on the viral multiplication and reduced the synthesis of viral specific mRNA and proteins. No inhibitory effect was observed when the cells were treated with sense oligonucleotide and oligonucleotide which contained unrelated sequences at concentrations from 1 to 10 microM. These results showed that antisense oligonucleotide against the leader RNA reduced the multiplication of positive-stranded RNA virus, MHV.

Animals↗