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PubMed1982

Organization and expression of the poxvirus genome.

Poxviruses comprise a large group of very complex animal DNA viruses which replicate in the cytoplasm of infected cells. Vaccinia virus, the most studied poxvirus, has a linear, double stranded DNA genome with an approximate molecular weight of 120 x 10(6) (180 kilobase pairs). The two strands of the DNA molecule are naturally cross-linked at both termini. In addition, the vaccinia virus genome contains very long inverted terminal repetitions of approximately 10 kilobase pairs which are further characterized by the presence of direct tandem repeats of a 70-base-pair sequence arranged in two blocks of 13 and 17 copies, respectively. A central region of the genome is highly conserved between different orthopoxviruses. In contrast, the ends are hypervariable and may contain extensive deletions and complex, symmetrical sequences rearrangements. Vaccinia virus gene expression is divided into two stages. Early in infection, RNA complementary to one half of one strand-equivalent of the genome is transcribed within subviral particles by the virion-associated RNA polymerase. Later in infection, after DNA replication, RNA complementary to one entire strand-equivalent is transcribed. RNA made late in infection is very heterogeneous in length and a large fraction of it contains self-complementary sequences. Late genes are clustered near the central region of the genome. Vaccinia virus mRNAs do not appear to be synthesized by a splicing mechanism.

Base Sequence↗

PubMed1981

Titration of integrated simian virus 40 DNA sequences, using highly radioactive, single-stranded DNA probes.

Nick-translated simian virus 40 (SV40) [32P]DNA fragments (greater than 2 X 10(8) cpm/micrograms) were resolved into early- and late-strand nucleic acid sequences by hybridization with asymmetric SV40 complementary RNA. Both single-stranded DNA fractions contained less than 0.5% self-complementary sequences; both included [32P]-DNA sequences that derived from all regions of the SV40 genome. In contrast to asymmetric SV40 complementary RNA, both single-stranded [32P]DNAs annealed to viral [3H]DNA at a rate characteristic of SV40 DNA reassociation. Kinetics of reassociation between the single-stranded [32P]DNAs indicated that the two fractions contain greater than 90% of the total nucleotide sequences comprising the SV40 genome. These preparations were used as hybridization probes to detect small amounts of viral DNA integrated into the chromosomes of Chinese hamster cells transformed by SV40. Under the conditions used for hybridization titrations in solution (i.e., 10- to 50-fold excess of radioactive probe), as little as 1 pg of integrated SV40 DNA sequence was assayed quantitatively. Among the transformed cells analyzed, three clones contained approximately one viral genome equivalent of SV40 DNA per diploid cell DNA complement; three other clones contained between 1.2 and 1.6 viral genome equivalents of SV40 DNA; and one clone contained somewhat more than two viral genome equivalents of SV40 DNA. Preliminary restriction endonuclease maps of the integrated SV40 DNAs indicated that four clones contained viral DNA sequences located at a single, clone-specific chromosomal site. In three clones, the SV40 DNA sequences were located at two distinct chromosomal sites.

Animals↗

PubMed1999

Corticotropin-releasing factor receptor expression in the pituitary of fetal sheep after lesion of the hypothalamic paraventricular nucleus.

Both the capacity of CRF to release ACTH and the number of binding sites for CRF in the anterior pituitary decline during the final weeks of gestation in fetal sheep. The present study examined regulation of pituitary CRF receptor expression by the hypothalamic paraventricular nucleus (PVN) during late gestation in fetal sheep. Bilateral radiofrequency lesions of the PVN (PVN-Lx; n = 4) or sham lesions (SHAM; n = 5) were performed in fetal sheep at 118-122 days of gestational age (dGA). Pituitary glands from PVN-Lx and SHAM fetuses were collected at 139-142 dGA (term, approximately 148 dGA). Dual-label in situ hybridization was performed using a digoxigenin-labeled ovine POMC complementary RNA, together with a 35S-labeled ovine CRF type I (CRF1) receptor complementary RNA, to localize and quantify CRF1 receptor mRNA in POMC-hybridizing cells. Binding of [125I]-ovine CRF was also examined in the fetal pituitary of both PVN-Lx and SHAM fetuses using in situ autoradiography. The hybridization signal for the CRF1 receptor mRNA was primarily restricted to POMC-expressing cells in the anterior pituitary of both PVN-Lx and SHAM fetuses; no hybridization signal for the CRF1 receptor was observed in the neurointermediate lobe (NIL) in either group. The hybridization signal for CRF1 receptor mRNA in anterior pituitary corticotropes of PVN-Lx fetuses was significantly lower in both the inferior and superior regions of the anterior pituitary, compared with SHAM fetuses (P < 0.05). In the inferior region of the anterior pituitary, the percentage of POMC-hybridizing cells containing CRF1 receptor hybridization signal was significantly greater in PVN-Lx (90+/-7%; mean +/- SEM), compared with SHAM (67+/-6%; P < 0.05) fetuses. No differences in the percentage of POMC cells containing CRF1 receptor hybridization signal were observed in the superior region of the anterior pituitary between PVN-Lx (89+/-8%) and SHAM (87+/-9%). Binding of [125I]-ovine CRF (oCRF) was significantly greater in anterior pituitaries of PVN-Lx (140+/-19 mean arbitrary densitometry U +/- SEM), compared with SHAM (73+/-23; P < 0.05) fetuses. For both PVN-Lx and SHAM fetuses, there were no differences within group in [125I]-oCRF binding between the inferior and superior regions of the anterior pituitary. A weak, but significant (P < 0.05), autoradiographic signal for [125I]-oCRF binding was observed in the NIL of both SHAM and PVN-Lx fetal sheep. The level of [125I]-oCRF binding was significantly lower in the NIL, compared with anterior pituitary, for both SHAM (P < 0.01) and PVN-Lx fetuses. There were no differences in [125I]-oCRF binding in the NIL between SHAM and PVN-Lx fetal sheep. Our findings support a role for the PVN in regulating anterior pituitary CRF1 receptor expression in the late-gestation sheep fetus.

Animals↗

PubMed1971

Mechanism of synthesis of vaccinia virus double-stranded ribonucleic acid in vivo and in vitro.

The synthesis of vaccinia virus double-stranded ribonucleic acid (RNA) in infected HeLa cells was sensitive to actinomycin D, suggesting that a deoxyribonucleic acid dependent reaction is involved. Some double-stranded RNA was made in the presence of cytosine arabinoside in infected cells. Double-stranded and complementary RNA were synthesized in vitro by using vaccinia cores. These two observations indicate that some of the double-stranded RNA is read from "early" genes. The double-stranded RNA synthesized in vitro had the same properties as that made in vivo. At least 70% of the double-stranded RNA made in vivo was in ribonuclease-resistant form prior to sodium dodecyl sulfate-phenol extraction. In addition, there was a complementary RNA in infected cells which could be converted to double-stranded RNA by annealing.

Centrifugation, Density Gradient↗

PubMed1990

Inhibition of flower pigmentation by antisense CHS genes: promoter and minimal sequence requirements for the antisense effect.

Introduction of a constitutive antisense full-length chalcone synthase (CHS) cDNA gene in petunia can result in an inhibition of flower pigmentation. We have evaluated some of the factors which may be important for the effectiveness of an antisense CHS gene. Antisense CHS genes encoding half-length or quarter-length RNA complementary to the 3' half of CHS mRNA are able to affect flower pigmentation, while a gene encoding RNA complementary to the 5' half of CHS mRNA did not show phenotypic effects in transgenic petunia plants. We demonstrate that the RNA encoded by the latter gene has a much lower average steady-state level in leaf tissue than the RNAs encoded by the other antisense gene constructs. We have compared the CaMV 35S and endogenous CHS promoter strengths and intrinsic stabilities of sense and antisense CHS RNAs. From the data we conclude that the constitutive antisense CHS genes are not likely to provide an excess of antisense RNA compared to the CHS mRNA derived from the endogenous genes. Effective inhibition of flower pigmentation is also observed when the antisense CHS gene is under control of the homologous CHS promoter. The results indicate that the mechanism of antisense inhibition cannot solely operate via RNA duplex formation between sense and antisense RNA.

Acyltransferases↗

PubMed1987

Nucleotide sequences of the PA and PB1 genes of B/Ann Arbor/1/66 virus: comparison with genes of B/Lee/40 and type A influenza viruses.

The complete sequences of the PA and PB1 genome RNA segments of B/Ann Arbor/1/66 virus have been determined. The PA vRNA is 2308 bases long. Its complementary RNA has a single open reading frame of 2187 bases, capable of encoding a PA protein of 726 amino acids with a molecular weight of 83,175 Da. The predicted PA polypeptide has an overall net charge of -7.5 at pH 7.0. The PB1 vRNA is 2369 bases long. Its complementary RNA has a single open reading frame of 2277 bases, capable of encoding a PB1 protein of 752 amino acids with a molecular weight of 84,332 Da. The predicted PB1 polypeptide has an overall net charge of +18.5 at pH 7.0. Sequence homology comparisons of the PA and PB1 polypeptides from B/Ann Arbor/1/66 virus to the PA and PB1 polypeptides of type A influenza virus reveal respective homologies of approximately 38 and 60%. This high cross-type homology (61%) was previously reported for the PB1 protein of B/Lee/40 virus (Kemdirim et al., 1986). The cross-type homology for the PA protein is similar to that of other non-polymerase proteins, but is substantially lower than that seen for the PB1 protein. Thus, the high cross-type homology that exists for the PB1 gene does not appear to be a characteristic of all polymerase genes.

Amino Acid Sequence↗

PubMed2002

Analysis of minimal promoter sequences for plus-strand synthesis by the Cucumber necrosis virus RNA-dependent RNA polymerase.

Tombusviruses are small, plus-sense, single-stranded RNA viruses of plants. A partially purified RNA-dependent RNA polymerase (RdRp) preparation of Cucumber necrosis virus (CNV), which is capable of de novo initiation of complementary RNA synthesis from either plus-strand or minus-strand templates, was used to dissect minimal promoter sequences for tombusviruses and their defective interfering (DI) RNAs. In vitro RdRp assay revealed that the core plus-strand initiation promoter included only the 3'-terminal 11 nucleotides. A hypothetical promoter-like sequence, which has been termed consensus sequence by Wu and White (1998, J. Virol. 72, 9897-9905), is recognized less efficiently by the CNV RdRp than the core plus-strand initiation promoter. The CNV RdRp can efficiently recognize the core plus-strand initiation promoter for a satellite RNA associated with the distantly related Turnip crinkle virus, while artificial AU- or GC-rich 3'-terminal sequences make poor templates in the in vitro assays. Comparison of the "strength" of minimal plus-strand and minus-strand initiation promoters reveals that the latter is almost twice as efficient in promoting complementary RNA synthesis. Template competition experiments, however, suggest that the minimal plus-strand initiation promoter makes an RNA template more competitive than the minimal minus-strand initiation promoter. Taken together, these results demonstrate that promoter recognition by the tombusvirus RdRp requires only short sequences present at the 3' end of templates.

Cucumis sativus↗

PubMed1992

Sense and antisense TGF beta 3 mRNA levels correlate with cardiac valve induction.

The formation of the valves in the heart is a spatially and temporally controlled process. A tissue interaction between the endothelium and its adjacent myocardium initiates the transformation of the endothelium into the mesenchymal precursors of the heart valve. One or more of the molecules implicated as critical for valve formation are members of the transforming growth factor beta family of molecules. Presented here is a spatial and temporal analysis of TGF beta 2 and TGF beta 3 in the chick heart during valve formation. We show that TGF beta 3 mRNA is concentrated in AV canal tissue where valve formation will occur, consistent with previous observations that TGF beta 3 production is critical during valve formation. Additionally, an RNA complementary to TGF beta 3 encoding mRNA is present in the heart. The temporally controlled appearance of RNA complementary to TGF beta 3 suggests that this molecule may play a role in the regulation of TGF beta 3 production in the heart.

Animals↗

PubMedDate not supplied

[Functional value of 980-1061 sequences of human 18S ribosomal RNA using complementary DNA probes].

Region 980-1061 in human 18S rRNA was chosen on the basis of our previous results indicating, that the cross-linking sites of alkylating mRNA analogs are located within this region. In the present study, we have used 10 DNA 15-mers complementary to various overlapping sequences within the 18S rRNA positions 980-1061. Their ability to bind selectively at the desired rRNA sequences was proved by hydrolysis of 18S rRNA within heteroduplexes with the corresponding probes by RNase H. Only four of the probes were able to bind to 40S subunits indicating, that the corresponding 18S rRNA sequences 980-994, 987-1001, 1025-1039 and 1032-1046 are exposed within the subunits. None of the probes inhibited tRNA-dependent binding of oligo(U) messengers to 40S subunits. Nevertheless, two probes (complementary to 18S rRNA sequences 987-1001 and 1025-1039) being covalently attached to 40S subunits, inhibited translation of poly(U) by human 80S ribosomes in a cell-free system. The binding of messenger trinucleotide in the complex pAUG.40S.Met-tRNA.eIF-2.GTP was strongly affected by the same oligomers. Thus 987-1001 and 1025-1039 18S rRNA sequences are supposed to be involved in interaction with mRNA in the course of translation.

Base Sequence↗

PubMed1979

Quantitative analysis of specific labelled RNA'S using DNA covalently linked to diazobenzyloxymethyl-paper.

Substantial amounts of DNA (at least 25 microgram per cm2) can be stably bound to diazobenzyloxymethyl (DBM)-paper. Complementary RNA will hybridize to the DNA paper almost completely in 24 hours. Using several different conditions of hybridization and washing, the background of RNA bound non-specifically is very low (between 0.01 and 0.02%) and the efficiency of hybridization is very high (75 to 50% of complementary RNA is bound and retained through the washing procedure). Because the DNA is bound to the paper convalently, it is retained through all the washing and elution steps, and the DNA papers can be re-used many times.

Chromatography, Affinity↗

PubMed1991

Hybridization properties of deoxyoligonucleotides containing anthraquinone pseudonucleosides.

Achiral pseudonucleosides bearing an anthraquinone moiety have been incorporated into deoxyoligonucleotides in internal, 3' and 5' positions. The ability of these modified deoxyoligonucleotides to hybridize to complementary RNA and DNA was investigated using Tm measurements. The anthraquinone was shown to enhance binding to a complementary RNA when linked to the 3' and 5' end. Pseudonucleoside substitutions on the 3' end of an oligonucleotide are also shown to confer serum stability to the oligonucleotide.

Anthraquinones↗

PubMed1981

Cytochemical hybridisation with fluorochrome-labelled RNA. III. Increased sensitivity by the use of anti-fluorescein antibodies.

A new method to localise specific DNA sequences in microscopic preparations by hybridocytochemistry using fluorochrome labelled complementary RNA has been described recently (Bauman et al. 1981). The present paper describes a procedure to increase the sensitivity of this method. RNA complementary to kinetoplasts DNA of Crithidia luciliae was labelled with fluorescein and hybridised with Sephadex beads to which kinetoplast DNA or heterologous DNA had been covalently bound as well as to Crithidia luciliae preparations. The fluorescein-labelled RNA was found to hybridize specifically with homologous DNA both on the beads and in the cells. The sensitivity of the hybrid detection could be increased by applying an indirect immunofluorescence reaction using rabbit antiserum raised against the hapten fluorescein as has been described for the amplification of a direct immunofluorescence reaction by Schmitz and Kampa (1979). The complete procedure resulted in an amplification of the original specific fluorescence both on the beads and in the cells. The increase was quantified by microfluorimetry. Several aspects of the immunocytochemical amplifying reaction were quantitatively investigated using Sephadex beads to which poly(A) or DNA was coupled and FITC-labelled poly(U) or cRNA was hybridised. A 5- to 10-fold amplification was obtained both in the beads and on the cell preparations. When the amplifying steps were repeated a proportional increase in background fluorescence was observed.

Crithidia↗

PubMed1980

An abundant cytoplasmic 7S RNA is complementary to the dominant interspersed middle repetitive DNA sequence family in the human genome.

Evidence is presented that a homogeneous cytoplasmic species known as 7S RNA is the only abundant RNA in uninfected HeLa cells which can form strong hybrids with the dominant family of middle repetitive DNA sequences in the human genome. These DNA sequences are known collectively as the Alu family, because most of them share a common Alu I restriction site. When purified 7S RNA was hybridized to three different genomic clones containing Alu family DNA sequences, a specific region (or regions) comprising at most half the RNA sequence was protected from mild digestion with T1 ribonuclease; moreover, the hybrids between 7S RNA and cloned Alu family DNA wer imperfect, since T1 RNAase was able to nick the protected 7S RNA sequences under conditions where a true RNA: DNA duplex would have been resistant. This suggests that 7S RNA is encoded either by a small subset of the 300,000 Alu family sequences in the human genome or by an entirely different family of genes. The sequence of 7S RNA has been highly conserved through recent evolution, and in both avian and murine cells the RNA is selectively incorporated into oncornavirus particles during productive infection. The cellular function of 7S RNA is unknown.

Cytoplasm↗

PubMed1972

Patterns of Simian Virus 40 DNA transcription after acute infection of permissive and nonpermissive cells.

Small amounts of fractionated, denatured, (32)P-labeled DNA from SV40 virus were incubated with a large excess of the complementary RNA of SV40 prepared in vitro with Escherichia coli RNA polymerase; the viral DNA strands were separated on hydroxyapatite columns. The RNA present in green monkey cells late in the lytic cycle reacted with 40-42% of the strand complementary to the in vitro complementary RNA (minus strand), and 60-64% of the opposite (plus) strand. "Early lytic" RNA failed to significantly interact with the plus strand, but formed stable duplex molecules with 35-39% of the minus strand. The RNA prepared from mouse embryo cells 24 hr after infection with SV40 combined with 35-38% of the minus strand and 60-62% of the plus strand. In all cases, the same regions of either the plus or minus strand appear to be transcribed in permissive and nonpermissive infections.

Animals↗

PubMed1973

125 I-labeled DNA-RNA hybrids in cytological preparations.

RNA complementary to bulk humanplacental DNA was synthesized in vitro both in the presence and absence of (3)H-labeled ribonucleotides. The (3)H-labeled RNA was used directly for hybridization to the DNA of human metaphase chromosomes, whereas the unlabeled complementary RNA was labeled chemically with (125)I before hybridization. A comparison of autoradiographs produced by either isotope revealed no qualitative differences in the chromosomal annealing sites of the same population of RNA molecules. Since (125)I-labeled nucleic acids give similar, if not identical, results as do (3)H-labeled nucleic acids in in situ hybridization experiments, their use should make possible the localization of genetic elements for which tritium labeling methods are either inadequate or not possible.

Autoradiography↗

PubMed1979

Characterization and transcription analysis of a cloned sequence derived from a major developmentally regulated mRNA of D. discoideum.

The plasmid pDd 812 contains a portion of a poly(A)+ RNA sequence isolated from developing cells of the cellular slime mold Dictyostelium discoideum (Williams and Lloyd, 1979). The poly(A)+ RNA complementary to this plasmid shows an increase in concentration during the first 4 hr of development followed by a decrease in concentration during the following 4 hr. This RNA is very abundant after 3-4 hr of development, constituting at least 2% of the poly(A)+ RNA population. In this study, we demonstrate that this poly(A)+ RNA is an mRNA sequence by translating the RNA complementary to pDd 812 in a heterologous system. The mRNA directs the synthesis of a major polypeptide of 33,000 daltons and a minor polypeptide of 31,000 daltons. We have used the plasmid DNA immobilized on filters to analyze the transcription of this RNA sequence in isolated nuclei. The amount of transcript synthesized in nuclei isolated at various stages of development which was complementary to pDd 812 changed in the same way as did the cytoplasmic concentration of this RNA--that is, maximal transcription occurred after 3-4 hr of development. Because this result was observed using labeling periods as short as 5 min, we believe that this change is unlikely to reflect a change in the rate of processing of RNA. We interpret these results to indicate that, at least in part, the control of the synthesis of this RNA is at the level of gene transcription.

Base Sequence↗

PubMed2001

Recovery of infectious Ebola virus from complementary DNA: RNA editing of the GP gene and viral cytotoxicity.

To study the mechanisms underlying the high pathogenicity of Ebola virus, we have established a system that allows the recovery of infectious virus from cloned cDNA and thus permits genetic manipulation. We created a mutant in which the editing site of the gene encoding envelope glycoprotein (GP) was eliminated. This mutant no longer expressed the nonstructural glycoprotein sGP. Synthesis of GP increased, but most of it accumulated in the endoplasmic reticulum as immature precursor. The mutant was significantly more cytotoxic than wild-type virus, indicating that cytotoxicity caused by GP is down-regulated by the virus through transcriptional RNA editing and expression of sGP.

Animals↗

PubMed2000

Transcription and replication of the influenza a virus genome.

The genome of influenza A virus consists of eight segments of negative-strand viral RNA (vRNA). During the replication cycle of the virus, the genomic vRNA is transcribed into positive-strand mRNA and complementary RNA (cRNA) in the cell nucleus. The promoter for the synthesis of mRNA molecules is located in a partially double-stranded RNA structure formed by the 5'- and 3'-terminal sequences of genomic vRNA segments. The virus encoded RNA-dependent RNA polymerase complex has to interact with both ends of the vRNA in order to generate capped RNA primers by endonucleolytic cleavage of cellular pre-mRNAs for the initiation of viral mRNA synthesis. Conserved sequence elements in the 5'-end, e.g. a polymerase binding site and a U(5-7) sequence are required for polyadenylation of virus-specific mRNAs. Polyadenylation occurs by reiterative copying of the U(5-7) sequence by the viral RNA polymerase, which is bound to the 5'end of the vRNA template. The U(5-7) sequence acts directly as a template for the poly(A)-tail. During the replication cycle of the virus, a "switch" from mRNA to cRNA synthesis occurs, but the mechanism by which this switch occurs remains unclear. The viral nucleoprotein and its interaction with the polymerase proteins and vRNA might play a role in this process. In contrast to transcription, the process of replication--the synthesis of cRNA and vRNA, which are known to occur in the absence of primers--is poorly understood.

Animals↗