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PubMed1991

Secondary structure of coliphage Q beta RNA. Analysis by electron microscopy.

The secondary structure of genomic RNA from the coliphage Q beta has been examined by electron microscopy in the presence of varying concentrations of spermidine using the Kleinschmidt spreading technique. The size and position of structural features that cover 70% of the viral genome have been mapped. The structural features that are visualized by electron microscopy in Q beta RNA are large. They range in size from 170 to 1600 nucleotides. A loop containing approximately 450 nucleotides is located at the 5' end of the RNA. It includes the initiation region for the viral maturation protein. A large hairpin containing approximately 1600 nucleotides is located in the center of the molecule. It is multibranched and includes most of the viral coat gene, the readthrough region of the A1 gene, and approximately one third of the viral replicase gene. Within the central hairpin, the initiation region for the viral replicase gene pairs with a region within the distal third of the viral coat gene. This structure may participate in the regulation of translational initiation of the viral replicase gene. Two structural variants of the central hairpin were observed. One of them brings the internal S and M viral replicase binding regions into juxtaposition. These observations suggest that the central hairpin may also participate in the regulation of translation of the viral coat gene. The secondary structures that are observed in Q beta RNA differ significantly from structures that we described previously in the genomic RNA of coliphage MS2 but are similar to structures we observed by electron microscopy in the related group B coliphage SP.

Coliphages↗

PubMed1994

Characterization of a point mutation in aspartylglucosaminidase gene: evidence for a readthrough of a translational stop codon.

We have identified a novel aspartylglucosaminuria (AGU) mutation in the second exon of the aspartylglucosaminidase (AGA) gene resulting in a lysosomal storage disease in a Puerto Rican pedigree. This T192-->A transversion causes replacement of Cys64 with a premature translational stop codon and the patients' fibroblasts exhibit dramatically decreased steady-state levels of AGA mRNA. Immunofluorescence analysis and analysis of immunoprecipitated metabolically labelled AGA polypeptides from patient fibroblasts unexpectedly revealed traces of normally sized inactive AGA precursor polypeptide instead of the predicted short polypeptide of 40 amino acids, thus demonstrating readthrough due to suppression of the premature translational stop codon. The translated AGA precursor is not processed further and remains inactive. The Cys64 substitution evidently disturbs the folding of the nascent polypeptide in the endoplasmic reticulum, thus preventing activation by proteolytic cleavage.

Amino Acid Sequence↗

PubMed1994

Evidence that ORF 1 and 2 are the only virus-encoded replicase genes of cymbidium ringspot tombusvirus.

The 33-kDa and its 92-kDa readthrough protein genes (open reading frames (ORFs) 1 and 2) of cymbidium ringspot tombusvirus (CyRSV) were introduced into Nicotiana benthamiana plants. Protoplasts derived from transgenic plants expressing the first two ORFs were inoculated with in vitro transcripts of defective interfering (DI) RNA. The efficient replication of input DI RNA, which is incapable of autonomous replication, confirmed the biological activity of the expressed viral replicase. In addition, in vitro transcripts of a genomic RNA deletion mutant clone encoding only ORFs 1 and 2 were able to replicate on their own and could support the replication of CyRSV DI RNAs in N. benthamiana protoplasts.

Amino Acid Sequence↗

PubMed1989

Biochemical and genetic analysis of Salmonella typhimurium and Escherichia coli mutants defective in specific incorporation of selenium into formate dehydrogenase and tRNAs.

Mutation of a single gene, referred to as selA1 in Salmonella typhimurium and as selD in Escherichia coli, results in the inability of these organisms to insert selenium specifically into the selenopolypeptides of formate dehydrogenase and into the 2-selenouridine residues of tRNAs. The mutation does not involve transport of selenite into the cell or reduction of selenite to selenide since both mutant strains synthesize selenocysteine and selenomethionine from added selenite and incorporate these selenoamino acids non-specifically into numerous proteins of the bacterial cells. Complementation of the mutation in S. typhimurium with the selD gene from E. coli indicates functional identity of the selA1 and selD genes. Although the selA1 gene maps at approximately 21 min on the S. typhimurium chromosome and the selD gene at approximately 38 min on the E. coli chromosome, only a single gene in wild-type S. typhimurium hybridized to the E. coli selD gene probe. Transformation of the mutant Salmonella strain with a plasmid bearing the E. coli selD gene restored formate dehydrogenase activity, 75Se incorporation into formate dehydrogenase seleno-polypeptides and [75Se]seleno-tRNA synthesis. Transformation with an additional plasmid carrying an E. coli formate dehydrogenase selenopolypeptide-lacZ gene fusion showed that the selD gene allowed readthrough of the UGA codon and synthesis of beta-galactosidase in the Salmonella mutant.

Aldehyde Oxidoreductases↗

PubMed1997

Readthrough activation of early adenovirus E1b gene transcription.

In cells productively infected with adenovirus type 5, transcription is not terminated between the E1a gene and the adjacent downstream E1b gene. Insertion of the mouse beta(maj)-globin transcription termination sequence (GGT) into the E1a coding region dramatically reduces early, but not late, E1b expression (E. Falck-Pedersen, J. Logan, T. Shenk, and J. E. Darnell, Jr., Cell 40:897-905, 1985). In the study described herein, we showed that base substitution mutations in the globin DNA that specifically relieved transcription termination also restored early E1b promoter activity in cis, establishing that maximal early E1b expression requires readthrough transcription originating from the adjacent upstream gene. To identify potential targets of readthrough activation, a series of recombinant viruses with double mutations was constructed. Each double-mutant virus strain had the transcription termination sequences in the first exon of E1a and a deletion within the transcription control region of E1b. Early E1b expression from the double-mutant strains was more defective than that from strains containing either mutation alone, indicating that the deleted regions (positions -362 to -35) are not the target for readthrough activation. Two findings suggested that a cis-dominant property of early viral templates is important for readthrough activation. First, the early E1b defect caused by the GGT insertion was not complemented in trans by factors present in late-infected cells. Second, restoration of E1b transcription at late times occurred concurrently with viral DNA replication. Readthrough activation may help convert virion DNA into a transcriptionally competent template prior to DNA replication and late transcription.

Adenovirus E1A Proteins↗

PubMed1992

Conservative system for dosage-dependent modulation of translational fidelity in eukaryotes.

Variations in dosage of some genes can alter the level of translational fidelity. The Saccharomyces cerevisiae genes that act as dosage-dependent suppressors and/or modulators of suppression, are the following: some tRNA genes (for example, tRNA(Gln)) inducing readthrough by mispairing; genes coding for either translational elongation factor or other proteins taking part in translation; and some genes of unknown function. We suggest that the SUP35 protein is a factor which may play a major role in balance-dependent regulation of translational fidelity. Homologues of this genes have been identified in other yeast genera (Pichia), green algae (Chlamydomonas) and various animals including man. No homologies have been found in the polychaeta (Nereis) or in insects (Drosophila). Rates of evolution differ for two separate parts of the genes; the N-terminal part, which is important for ambiguous translation in Saccharomyces, is markedly variable in the organisms tested. However, the C-terminal part which is required for yeast viability has a common origin but a separate evolution from that of the EF-Tu protein family.

Animals↗

PubMed2003

Local character of readthrough activation in adenovirus type 5 early region 1 transcription control.

Wild-type early activity of the adenovirus 5 E1b gene promoter requires readthrough transcription originating from the adjacent upstream E1a gene. This unusual mode of viral transcription activation was identified by genetic manipulation of the mouse beta(maj)-globin gene transcription termination sequence (GGT) inserted into the E1a gene. To facilitate further study of the mechanism of readthrough activation, the activities of GGT and a composite termination sequence CT were tested in recombinant adenoviruses containing luciferase reporters driven by the E1b promoter. There was a strict correlation between readthrough and substantial downstream gene expression, indicating that interference with downstream transcription was not a unique property of GGT. Blockage of readthrough transcription of E1a had no apparent effect on early expression of the major late promoter, the next active promoter downstream of E1b. A test for epistatic interaction between termination sequence insertions and E1a enhancer mutations suggested that readthrough activation and E1a enhancer activation of the E1b promoter are mechanistically distinct. In addition, substitution of the human cytomegalovirus major immediate-early promoter for the E1b promoter suppressed the requirement for readthrough. These results suggest that readthrough activation is a "local" effect of a direct interaction between the invading transcription elongation complex and the E1b promoter. DNase I hypersensitivity footprinting provided evidence that this interaction altered an extensive E1b promoter DNA-protein complex that was assembled in the absence of readthrough transcription.

Adenovirus E1A Proteins↗

PubMed1997

Isolation and characterization of vesicular stomatitis virus PoIR revertants: polymerase readthrough of the leader-N gene junction is linked to an ATP-dependent function.

The switch from transcription to replication of the VSV genome is coupled to assembly of nascent chains and involves an unspecified change in the P-L polymerase complex when it reaches the leader-N gene junction. PoIR VSV mutants, in contrast to wild-type virus, read through this first gene junction at high frequency without concurrent assembly, and they show altered ATP requirements for transcription in vitro. The mutation(s) responsible for the poIR phenotype segregates to the N-RNA template fraction. We report here that both poIR1 and poIR2 mutants display severe growth restriction in mouse L cells but not in BHK cells. Four of six poIR1 revertant viruses, originating from rare plaques on L cells, showed wild-type characteristics for growth, readthrough of leader-N gene junction, and ATP utilization, while two showed partial and quantitatively parallel coreversion of all properties. Sequence analysis of N and P genes of poIR mutants and revertants provided proof that a single mutation in the N protein, Arg179 to His, is responsible for the poIR phenotype. PoIR1, but not poIR2, also displayed a phenotypically silent GA-to-GG change in the N-P intergenic dinucleotide sequence Five of six revertants retained the poIR1 N protein mutation and showed no change in their P gene. We conclude that the L protein likely contains second-site suppressors of the poIR phenotype, and we propose that the switch from transcription to replication is modulated by an ATP-dependent interaction between the template-associated N protein and the L subunit of the P L polymerase complex.

Adenosine Triphosphate↗

PubMed1991

Transcriptional interference in transgenic plants.

When a promoterless marker gene is transformed into the plant genome using the Agrobacterium vector system, on average 30% of the T-DNA inserts produce gene fusions. This suggests that the T-DNA is preferentially integrated into transcribed regions. Here, we proposed that this transcriptional activity is responsible for some of the variation in expression frequently observed among independent transformants. Using hybrid gene constructions, we show that transcriptional readthrough into a downstream gene with opposite orientation substantially reduces expression of this gene both in transient expression and in transgenic plants. Furthermore, a poly(A) signal/terminator can block readthrough and restore the expression of the gene. Finally, enzymatic analysis of calli suggests that less variation in neomycin phosphotransferase II synthesis is observed when the gene is separated from plant DNA by promoter and terminator elements.

Agrobacterium tumefaciens↗

PubMed1996

Local and distant sequences are required for efficient readthrough of the barley yellow dwarf virus PAV coat protein gene stop codon.

Many viruses use stop codon readthrough as a strategy to produce extended coat or replicase proteins. The stop codon of the barley yellow dwarf virus (PAV serotype) coat protein gene is read through at a low rate. This produces an extended polypeptide which becomes part of the virion. We have analyzed the cis-acting sequences in the barley yellow dwarf virus PAV genome required for this programmed readthrough in vitro in wheat germ extracts and reticulocyte lysates and in vivo in oat protoplasts. Two regions 3' to the stop codon were required. Deletion of sections containing the first 5 of the 16 CCN NNN repeats located 3' of the stop codon greatly reduced readthrough in vitro and in vivo. Surprisingly, readthrough also required a second, more distal element that is located 697 to 758 bases 3' of the stop codon within the readthrough open reading frame. This element also functioned in vivo in oat protoplasts when placed more than 2 kb from the coat protein gene stop in the untranslated region following a GUS reporter gene. This is the first report of a long-range readthrough signal in viruses.

Avena↗

PubMed1989

Transcriptional regulation of F plasmid gene srnB: rifampicin-promoted in vitro readthrough of a terminator in the leader region.

srnB is an F-plasmid encoded gene, otherwise silent, whose expression is induced by added rifampicin, leading to the release of cellular Mg2+ and degradation of stable RNA. In the absence of rifampicin, transcripts from the srnB gene were relatively short. S1 nuclease mapping revealed that the short mRNA species terminated within the leader, at the 3' end of a potential stem-and-loop structure. A deletion in the stem-loop resulted in constitutive synthesis of the mRNA that extended beyond the termination site into the structural gene. Even with the wild-type gene, transcription continued beyond the terminator sequence in the presence of added rifampicin. Most of the transcripts synthesized in the presence of rifampicin were long enough to encode the srnB protein. We hypothesize from these results that RNA polymerase associated with rifampicin can read through the terminator to induce srnB expression.

Amino Acid Sequence↗

PubMed1990

A short, highly repetitive element in intron -1 of the human c-Ha-ras gene acts as a block to transcriptional readthrough by a viral promoter.

We have identified a short, highly repetitive element within intron -1 of the human c-Ha-ras gene. This element was found to be transcribed in both orientations and to be homologous to heterogeneous nonpolyadenylated transcripts. The repetitive element blocked transcriptional readthrough from a strong upstream viral promoter but allowed unimpaired readthrough from the c-Has-ras promoter. We suggest that it may serve to prevent excessive transcription into the coding region of the gene under such circumstances as viral insertion.

Cell Line↗

PubMed1986

Glutamine starvation of murine leukaemia virus-infected cells inhibits the readthrough of the gag-pol genes and proteolytic processing of the gag polyprotein.

The production of Moloney murine leukaemia virus from chronically infected cells was inhibited after starvation of glutamine. While the rate of synthesis of the precursor of the core proteins, Pr65gag, was not affected in the starved cells, its proteolytic processing was blocked. Pulse-chase experiments indicated that glutamine was required during the synthesis of Pr65gag to facilitate its subsequent processing. In addition, the synthesis of Pr200gag-pol, the precursor of the protease, reverse transcriptase and endonuclease, was inhibited in the glutamine-starved cells. Starvation for other essential amino acids such as tyrosine and isoleucine affected neither the synthesis nor the processing of the virus proteins. These results suggest that the readthrough mechanism which enables synthesis of the Pr200gag-pol polyprotein is modulated in the chronically infected cells by glutamine levels. Since the viral protease is part of the pol gene, its synthesis may be inhibited in the glutamine-starved cells and Pr65gag is therefore not processed.

Animals↗

PubMed1989

Translational readthrough of the murine leukemia virus gag gene amber codon does not require virus-induced alteration of tRNA.

An in vitro system to assay translational readthrough of the UAG termination codon at the murine leukemia virus (MuLV) gag-pol junction was developed by using rabbit reticulocyte lysates programmed by SP6-generated Moloney MuLV gag-pol mRNA. Under conditions in which the suppressor activity of the lysate was dependent on addition of tRNA, it could be shown that readthrough synthesis was stimulated to approximately the same extent by equivalent amounts of tRNA from MuLV-infected and uninfected NIH 3T3 cells. Analysis of glutamine tRNA, which mediates suppression in vivo, showed that the level of glutamine acceptor activity and the chromatographic profile of glutamine isoacceptors were unchanged following virus infection. On the basis of these results, we conclude that the suppressor tRNA occurs normally within the tRNA population of uninfected cells and need not be induced in response to virus infection.

Animals↗

PubMed1978

Studies on the control region of the bipolar argECBH operon of Escherichia coli. I. Effect of regulatory mutations and IS2 insertions.

Several mutations affecting the control or the potential of gene expression in the argECBH bipolar operon have been characterized by enzyme assays, genetic mapping, dominance tests and pulse labelled RNA determinations. None of the mutations involves DNA rearrangements detectable by heteroduplex analysis (Charlier et al., 1978). Partially constitutive transcription of both argE and argCBH has been observed in mutant L10 while constitutive argE transcription and normal argCBH control characterize mutants L9, LL13 and LL2. The control region thus appears to contain two overlapping operators, as suggested previously (Elseviers et al., 1972). Two mutants (L2, LL1) and strain 6-8 from Bretscher and Baumberg (1976) display an increase in acetylornithinase specific activity (argE product) without concommittant increased argE transcription. In addition, they exhibit a decreased argCBH transcription. It is suggested that in these organisms, argE translation and argCBH transcription may be affected by the same genetic event; this explanation is compatible with present working hypothesis for the structure of the control region. An interpretation in terms of messenger attenuation also appears possible. From the properties of two strains harbouring an IS2 insertion in the control region (Charlier et al., 1978) the following conclusion may be drawn: 1. When inserted in orientation I close to the proximal end of a silent gene IS2 appears to promote a low but detectable transcription readthrough into that gene. 2. Insertion of an IS2 element in orientation II close to a neighbouring gene is not a sufficient condition to express that gene at a high rate. The properties of the two insertions appear compatible with the structure proposed for the control region.

Acetyltransferases↗

PubMed1986

Transcriptional map of the measles virus genome.

We have constructed a recombinant DNA library of the measles virus genome and identified gene-specific clones containing sequences coding for portions of each of the six viral structural proteins, as well as clones coding for intercistronic sequences. By Northern blotting, we could order the clones according to the pattern of individual gene-specific and readthrough mRNAs. Clones corresponding to the N, P/C and M genes were identified by correlation of the mRNAs with their in vitro translation products; clones corresponding to the H, F and L genes were identified by indirect evidence. The results indicated that the gene order in measles virus is that of a typical paramyxovirus (3'-N,P/C,M,F,H,L-5'), but that the M and F transcripts each contain 1.5 times the coding capacity needed for synthesis of these proteins in vivo.

Cloning, Molecular↗

PubMed1995

Immunodetection, expression strategy and complementation of turnip crinkle virus p28 and p88 replication components.

The plus-sense RNA genome of turnip crinkle virus (TCV) encodes at its 5' end a 28-kDa protein of unspecified function. Readthrough suppression of the p28 stop codon allows for the production of an 88-kDa product which is required for genome replication. Immunological analysis of the expression of p28 and p88 demonstrated that: (i) the genome directs the synthesis of polypeptides of approximately 28 and 88 kDa, (ii) the 88-kDa protein is immunologically related to p28, consistent with p88 being a readthrough product, and (iii) p28, but not p88, is detectable in vivo. An in vivo assay, in which readthrough is linked to the expression of a beta-glucuronidase reporter gene, showed that readthrough of the p28 amber stop codon occurs with an efficiency of approximately 1%. A similar efficiency of readthrough was observed when an altered context from the nonviable TCV mutant, mA2, containing a disrupted secondary structure (FfFa) spanning the p28 termination codon, was tested. This result suggests that the defective phenotype of mA2 is likely not linked to an alteration in readthrough efficiency. Additional studies demonstrated that complementation occurs in coinoculations with two nonviable TCV mutants, RT and APA, which are unable to express either p28 or p88, respectively. This result verifies that p28 is essential for TCV genome replication and provides the first definitive evidence for the role of a 5'-proximal open reading frame for any member of the family Tombusviridae.

Base Sequence↗

PubMed2003

Analysis of whole-genome microarray replicates using mixed models.

MOTIVATION: Microarray experiments are inherently noisy. Replication is the key to estimating realistic fold-changes despite such noise. In the analysis of the various sources of noise the dependency structure of the replication needs to be taken into account. RESULTS: We analyzed replicate data sets from a Mycobacterium tuberculosis trcS mutant in order to identify differentially expressed genes and suggest new methods for filtering and normalizing raw array data and for imputing missing values. Mixed ANOVA models are applied to quantify the various sources of error. Such analysis also allows us to determine the optimal number of samples and arrays. Significance values for differential expression are obtained by a hierarchical bootstrapping scheme on scaled residuals. Four highly upregulated genes, including bfrB, were analyzed further. We observed an artefact, where transcriptional readthrough from these genes led to apparent upregulation of adjacent genes. AVAILABILITY: All methods and data discussed are available in the package YASMAhttp://www.cryst.bbk.ac.uk/wernisch/yasma.html for the statistical data analysis system R (http://www.R-project.org).

Algorithms↗