Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Readthrough gene”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 505 records · Page 28Linked to original sources
PubMed1997

Examination of the function of two kelch proteins generated by stop codon suppression.

The Drosophila kelch gene produces a single transcript with a UGA stop codon separating two open reading frames (ORF1 and ORF2). From the transcript, 76 kDa ORF1 and 160 kDa full-length (ORF1 + ORF2) proteins are made. The expression of these two proteins is regulated in a tissue-specific manner causing the ratio of full-length to ORF1 protein to vary in different tissues. The only detected defect for kelch mutants is female sterility, and kelch protein is localized to the ovarian ring canals. kelch mutant ring canals are disorganized and have partly occluded lumens, causing a failure to transport cytoplasm. ORF1 and full-length kelch proteins co-sediment with ring canals suggesting that both proteins are found in the ring canals. Transgenetic analysis reveals that ORF1 kelch protein is sufficient to rescue ring canal morphology and fertility. In addition, we have mutated the UGA stop codon to a UAA stop codon and to three sense codons that allow constitutive readthrough. Analysis of these mutants reveals that a full-length kelch protein can partially compensate for the loss of endogenous kelch, but the residue included at the stop codon is critical for function. Finally, these studies suggest that the mechanism of stop codon suppression of kelch is by tRNA suppression.

Alanine↗
PubMed1990

The block to transcription elongation is promoter dependent in normal and Burkitt's lymphoma c-myc alleles.

Aberrant c-myc expression patterns occur in human Burkitt's lymphoma cells, which consistently exhibit c-myc chromosomal translocations, mutations within and flanking the translocated allele, a loss of the block to transcription elongation in exon 1, and a promoter shift to use of the upstream P1 promoter. To define the mechanism responsible for the loss of transcription elongation blockage and resulting c-myc deregulation in Burkitt's lymphoma, we analyzed transcription patterns after transfer of normal and Burkitt's lymphoma c-myc alleles into murine cells and Xenopus oocyte germinal vesicles. We have determined that although the mutations within and surrounding several Burkitt's lymphoma c-myc alleles are not sufficient, in themselves, to abrogate the transcription elongation block, transcription initiation from the P2 promoter may be necessary to obtain the block to transcription elongation. To test directly the role of c-myc promoters in programming transcription elongation blockage, we analyzed transcription patterns from in vitro mutagenized c-myc genes containing deletions of either the P1 or P2 promoter. These data confirm that P1-initiated c-myc transcripts do not terminate at discrete sites near the 3' end of exon 1, whereas P2-initiated transcripts either terminate or read through the transcription block signals. Therefore, overexpression and/or constitutive expression from the c-myc P1 promoter may contribute to increased readthrough transcription in Burkitt's lymphoma cells and, hence, to aberrant expression patterns or levels of c-myc steady-state transcripts. In addition, the ability of normal cells to modulate c-myc P2-initiated transcription to either read through or to block elongation provides a fine control mechanism over c-myc steady-state RNA levels.

Alleles↗
PubMed2006

Glucose uptake pathway-specific regulation of synthesis of neotrehalosadiamine, a novel autoinducer produced in Bacillus subtilis.

Neotrehalosadiamine (3,3'-diamino-3,3'-dideoxy-alpha,beta-trehalose; NTD) is an amino-sugar antibiotic produced by several Bacillus species that functions as an autoinducer by activating its own biosynthetic operon, ntdABC. We previously reported that the introduction of a certain rpoB mutation (rpoB5) into Bacillus subtilis enables the cells to overproduce NTD. B. subtilis mini-Tn10 transposant libraries have been screened for genes that affect NTD production. Inactivation of ccpA, which encodes a major transcriptional regulator of carbon catabolite regulation, markedly reduced NTD production. By contrast, inactivation of glcP, which is situated just downstream of ntdABC and encodes a glucose/mannose:H(+) symport permease, stimulated NTD production. Overexpression of glcP led to the repression of ntdABC expression (and thus NTD production) in response to GlcP-mediated glucose uptake. These results suggest that CcpA-mediated catabolite activation of ntdABC expression occurs in response to the increase of the in vivo concentration of fructose-1,6-bisphosphate via glucose-6-phosphate and that GlcP-mediated glucose repression of ntdABC expression occurs in association with the increase of the in vivo concentration of unphosphorylated glucose. In addition, Northern analysis showed that glcP is transcribed from the ntdABC promoter through transcription readthrough at the ntdABC transcription terminator site, which enables NTD to function as a modulator of glucose uptake through the stimulation of ntdABC-glcP transcription, even in wild-type (rpoB(+)) cells. A trace amount (0.5 to 3 mug/ml) of NTD was sufficient to ensure expression of glcP, thus demonstrating the physiological role of "antibiotic" in the producing bacteria by functioning as an autoinducer for glucose uptake modulation.

Bacillus subtilis↗
PubMed2005

Comparison of the beet necrotic yellow vein virus P75 nucleotide sequences of Belgian type A and type B sources.

RNA-2 P75 open reading frame sequencing was conducted on BNYVV-infected sugar beet plants originating from 10 Belgian sources and was compared with sequences originating from France, Kazakhstan, Japan and China. This allowed the characterization of types A and B in Belgium. Sequence analysis confirmed that type A is intermediate between types P and B. The analysis of nucleotide sequences of the capsid protein epitopes and of different motifs involved in the transmission of the virus to the plant by Polymyxa betae reveals that they are well conserved among the different sequences analysed. In silico analysis showed that a potential dimerization/dsRNA-binding domain is present on P75. Finally, in order to compare the different BNYVV types, a real-time quantitative (RT)-PCR targeting the readthrough domain of P75 was developed and assessed over a period of 40 days on BNYVV susceptible sugar beet cv. Cadyx.

Amino Acid Sequence↗
PubMed1999

Nus A is involved in transcriptional termination on lambda tI.

The transcriptional terminator tI generates the 3'end of the integrase (int) gene transcript that is read from the lambda PI promoter in lambda phage. We have studied the factors that affect transcription termination in vitro and in vivo at the lambda tI terminator. In vitro transcriptional studies showed that tI is about 80% efficient in the presence of purified NusA protein, whereas it is only about 50% efficient in its absence. In vivo studies, where the readthrough transcript of lambda tI was measured by quantitative dot blot analysis, gave about 80% efficiency in wild-type strains, but only 60% in the nusA1 mutant strain at non-permissive temperatures. These results support the idea that termination at lambda tI in vivo involves interaction with the NusA factor.

Bacterial Proteins↗
PubMed2026

Small molecule inhibition of CPSF3 may impact R-loop distribution and abundance.

R-loops are three-stranded nucleic acid structures consisting of an RNA/DNA hybrid and a displaced strand of DNA. These structures have been implicated in a variety of regulatory cellular processes. Their untimed or excess accumulation, however, can cause genomic instability and induce DNA damage. Most R-loops form co-transcriptionally when the nascent transcript reanneals to unwound DNA duplex. Changes in transcription rates have the potential to impact R-loop formation, and compounds that modulate R-loop formation would be useful molecular tools and therapeutic leads. Cleavage and Polyadenylation Specific Factor 3 (CPSF3) recognizes the pre-mRNA 3' cleavage site, cleaves the transcript prior to polyadenylation, and has been linked to R-loop formation. Inhibition of CPSF3 has been found to induce transcriptional readthrough and cell proliferation defects. A previous report suggested that inhibition of CPSF3 with a small molecule causes a global increase in R-loop formation. Here, we test the impact of YT-II-100, a novel inhibitor of CPSF3. We find that addition of YT-II-100 increases global R-loop formation but does not change R-loop formation at specific genes that are normally used as positive controls for R-loop formation. We performed parallel assays using a previously reported compound, JTE-607, and observed similar results. Our data emphasize the need for cautious interpretation of experiments using JTE-607 and YT-II-100. There may be different mechanisms of R-loop formation depending on gene loci, where the control of R-loop formation by agonists at certain genes may differ from the trends observed for impacts on global R-loop formation.

R-Loop Structures↗
PubMed2003

The T box and S box transcription termination control systems.

The T box and S box transcription termination control systems are widely used for control of gene expression in Gram-positive bacteria, but are rare in Gram-negative organisms. Both of these systems can be recognized in genomic data because of high conservation of primary sequence and structural elements. The T box system regulates a variety of amino acid-related genes, while the S box system is dedicated to genes involved in methionine metabolism. While both systems involve gene regulation at the level of premature termination of transcription, the molecular mechanisms employed are very different. In the T box system, expression is induced by stabilization of an antiterminator structure in the leader by interaction with the cognate uncharged tRNA; this prevents formation of the competing terminator helix, allowing synthesis of the full-length mRNA. Disruption of conserved leader features results in loss of readthrough. In the S box system, the antiterminator form of the leader is the more stable form. A competing anti-antiterminator must be stabilized by an unknown factor during growth in methionine to prevent formation of the antiterminator, thereby allowing formation of the terminator helix. Disruption of conserved leader elements results in constitutive expression.

Base Sequence↗
PubMed1996

Cascade regulation of the toluene-3-monooxygenase operon (tbuA1UBVA2C) of Burkholderia pickettii PKO1: role of the tbuA1 promoter (PtbuA1) in the expression of its cognate activator, TbuT.

Burkholderia pickettii PKO1 metabolizes toluene and benzene via a chromosomally encoded toluene-3-monooxygenase pathway. Expression of the toluene-3-monooxygenase operon (tbuA1UBVA2C) is activated by the regulator, TbuT, in the presence of toluene. We have identified the TbuT coding region downstream of the toluene-3-monooxygenase structural genes by nucleotide sequence analysis and have shown that although TbuT is similar to XylR and DmpR, two members of the NtrC family of transcriptional activators which control toluene-xylene and (methyl)phenol catabolism, respectively, it is significantly different in the domain associated with effector specificity. Using a tbuA1-lacZ fusion reporter system, we determined that TbuT is activated not only by aromatic effectors but also the chlorinated aliphatic hydrocarbon trichloroethylene. Expression of tbuT and that of the tbuA1UBVA2C operon were found to be linked by readthrough transcription of tbuT from the toluene-3-monooxygenase promoter. As a result, transcription of tbuT is low when the toluene-3-monooxygenase operon is uninduced and high when expression of tbuA1UBVA2C is induced by toluene. Thus, the toluene-3-monooxygenase promoter drives the cascade expression of both the toluene-3-monooxygenase operon and tbuT, resulting in a positive feedback circuit. Examination of the nucleotide sequence upstream of the toluene-3-monooxygenase operon for promoter-like sequences revealed a -24 TGGC, -12 TTGC sequence, characteristic of sigma54 (rpoN)-dependent promoters. Primer extension and tbuA1-lacZ fusion analyses demonstrated that this -24, -12 promoter sequence, referred to as PtbuA1, was the toluene-3-monooxygenase promoter. Upstream of PtbuA1, a DNA region with dyad symmetry exhibited homology with the XylR-binding site present upstream of the Pu promoter. Deletions within this DNA sequence resulted in complete loss of expression from PtbuA1, suggesting that this region may serve as the TbuT-binding site.

Amino Acid Sequence↗
PubMed1989

Regulation of expression of the Pasteurella haemolytica leukotoxin determinant.

The Pasteurella haemolytica leukotoxin determinant is composed of four contiguous genes encoded on the same DNA strand and denoted lktCABD, in the order of their genetic organization. To gain a better understanding of the expression and regulation of the leukotoxin, the transcripts and promoters of the lkt determinant were mapped. Northern (RNA) blot analysis revealed two sets of transcripts. One set was 3.7 and 3.4 kilobases long, encoded lktCA, and comprised approximately 90% of the transcripts, whereas the other set was 7.4 and 7.1 kilobases long and encoded lktCABD. Two promoters were present, and each had features similar to the Escherichia coli consensus promoter sequences. Both promoters were located upstream from lktC; they were separated by 258 base pairs, as mapped by primer extension analysis. These results suggest a mechanism of expression similar to that of the related E. coli hemolysin. Transcription initiated upstream from lktC at either promoter and continued through lktC and lktA to a rho-independent transcriptional termination signal in the lktA-lktB intercistronic region. This signal attenuated expression by terminating 90% of transcription to generate the 3.7- and 3.4-kilobase lktCA transcripts. The remaining readthrough transcription generated full-length 7.4- and 7.1-kilobase lktCABD transcripts. Expression of the leukotoxin was greatly reduced by growth at 30 degrees C, pH 6.5, and Fe2+ limitation. These conditions also modulated the expression of a number of other secreted proteins, which suggests that all of these secreted proteins are controlled by the same regulatory mechanism.

Bacterial Toxins↗
PubMed1996

Ribosomal scanning past the primary initiation codon as a mechanism for expression of CTL epitopes encoded in alternative reading frames.

An increasing amount of evidence has shown that epitopes restricted to MHC class I molecules and recognized by CTL need not be encoded in a primary open reading frame (ORF). Such epitopes have been demonstrated after stop codons, in alternative reading frames (RF) and within introns. We have used a series of frameshifts (FS) introduced into the Influenza A/PR/8 /34 nucleoprotein (NP) gene to confirm the previous in vitro observations of cryptic epitope expression, and show that they are sufficiently expressed to prime immune responses in vivo. This presentation is not due to sub-dominant epitopes, transcription from cryptic promoters beyond the point of the FS, or internal initiation of translation. By introducing additional mutations to the construct exhibiting the most potent presentation, we have identified initiation codon readthrough (termed scanthrough here, where the scanning ribosome bypasses the conventional initiation codon, initiating translation further downstream) as the likely mechanism of epitope production. Further mutational analysis demonstrated that, while it should operate during the expression of wild-type (WT) protein, scanthrough does not provide a major source of processing substrate in our system. These findings suggest (i) that the full array of self- and pathogen-derived epitopes available during thymic selection and infection has not been fully appreciated and (ii) that cryptic epitope expression should be considered when the specificity of a CTL response cannot be identified or in therapeutic situations when conventional CTL targets are limited, as may be the case with latent viral infections and transformed cells. Finally, initiation codon readthrough provides a plausible explanation for the presentation of exocytic proteins by MHC class I molecules.

Animals↗
PubMed1989

Features of the rho-dependent transcription termination polar element within the hisG cistron of Salmonella typhimurium.

Previous genetic analysis showed that the polar effects of mutations in the hisG cistron of Salmonella typhimurium are dependent on the presence of a single putative transcription termination element within the hisG gene. In fact, all proximal mutations causing translation termination are strongly polar, whereas distal ones are not. The element was mapped by isolating mutations able to relieve the polar phenotype, and they were found to be small deletions in the region downstream of the translational stop codon (M. S. Ciampi and J. R. Roth, Genetics 118:193-202, 1988). In this study, we analyzed the his-specific RNAs synthesized in vivo in different strains harboring the polar frameshift hisG2148 mutation. The nature of the polarity effects is clearly transcriptional, since shorter RNA molecules were produced. When the hisG2148 mutation was transferred in a rho background or in strains harboring the small distal deletions, an increase in readthrough transcription was observed. The transcriptional termination element was characterized in more detail by performing high-resolution S1 nuclease mapping experiments. This analysis showed that (i) termination or exonucleolytic degradation following termination produced transcripts with heterogeneous 3' ends; (ii) this process is dependent on the transcription termination factor Rho, since relief of termination occurs in a rho background; and (iii) the element appears to function as a transcription terminator, at least to some extent, even in the course of active translation of the hisG cistron.

Amino Acid Sequence↗
PubMed1997

In vitro interactions of the aphid endosymbiotic SymL chaperonin with barley yellow dwarf virus.

Barley yellow dwarf virus (BYDV)-vector relationships suggest that there are specific interactions between BYDV virions and the aphid's cellular components. However, little is known about vector factors that mediate virion recognition, cellular trafficking, and accumulation within the aphid. Symbionins are molecular chaperonins produced by intracellular endosymbiotic bacteria and are the most abundant proteins found in aphids. To elucidate the potential role of symbionins in BYDV transmission, we have isolated and characterized two new symbionin symL genes encoded by the endosymbionts which are harbored by the BYDV aphid vectors Rhopalosiphum padi and Sitobion avenae. Endosymbiont symL-encoded proteins have extensive homology with the pea aphid SymL and Escherichia coli GroEL chaperonin. Recombinant and native SymL proteins can be assembled into oligomeric complexes which are similar to the GroEL oligomer. R. padi SymL protein demonstrates an in vitro binding affinity for BYDV and its recombinant readthrough polypeptide. In contrast to the R. padi SymL, the closely related GroEL does not exhibit a significant binding affinity either for BYDV or for its recombinant readthrough polypeptide. Comparative sequence analysis between SymL and GroEL was used to identify potential SymL-BYDV binding sites. Affinity binding of SymL to BYDV in vitro suggests a potential involvement of endosymbiotic chaperonins in interactions with virions during their trafficking through the aphid.

Amino Acid Sequence↗
PubMed1985

Nucleotide sequences of the 1B and 1C nonstructural protein mRNAs of human respiratory syncytial virus.

The genes encoding the 1C and 1B mRNAs of human respiratory syncytial (RS) virus are first in the order of viral transcription and encode nonstructural (NS) proteins of approximate molecular weights 14,000 and 11,000, respectively, estimated by gel electrophoresis. The complete nucleotide sequences of the 1C and 1B mRNAs determined from several full-length cDNA clones are described. The 1C and 1B mRNAs contain 528 and 499 nucleotides, respectively, exclusive of poly(A), and encode proteins of 139 and 124 amino acids. The calculated molecular weights of the predicted NS1C and NS1B proteins are 15,567 and 14,674, respectively. Both mRNA sequences contain the 5'-terminal sequence, 5' GGGGCAAAU . . . , and the 3'-terminal sequence, 5' . . . AGUAUA(N)1-4-poly(A), that were identified previously as conserved among six other RS viral mRNAs. In addition, a dicistronic readthrough RNA having the general structure 5' 1C mRNA-intergenic sequence-1B mRNA 3' was identified by dideoxynucleotide sequencing of intracellular poly(A)+ RNA using a DNA primer derived from a 1B-cDNA clone. In the dicistronic RNA, the nucleotide sequences of the 1C and 1B cistrons are separated by, in mRNA sense, four A residues and the intergenic sequence 5' . . . CUUAACAGAAGACAAAAAN . . . 3' (N represents unidentified nucleotide). The significance of these sequences is discussed.

Base Sequence↗
PubMed1998

Use of a coupled transcriptional system for consistent overexpression and purification of UDG-Ugi complex and Ugi from Escherichia coli.

We have designed a novel coupled transcriptional construct wherein Escherichia coli uracil DNA glycosylase (UDG) and Bacillus subtilis phage PBS-2 encoded uracil DNA glycosylase inhibitor protein (Ugi) genes were cloned in tandem, downstream of an inducible promoter (Ptrc). Use of this bicistronic operon has allowed purification of large amounts of UDG-Ugi complex formed in vivo. The system has also been exploited for purification of large amounts of Ugi. While establishing the expression system, one of the constructs showed detectable suppression of UAG termination codon and resulted in accumulation of a minor population of a putative readthrough polypeptide corresponding to UDG. We discuss the likely occurrence of such a phenomenon in overproduction of other recombinant proteins. Finally, the usefulness of the operon construct in convenient mutational analysis to study the mechanism of UDG-Ugi interaction is also discussed.

Bacillus Phages↗
PubMed1988

Sequence and organization of barley yellow dwarf virus genomic RNA.

The nucleotide sequence of the genomic RNA of barley yellow dwarf virus, PAV serotype was determined, except for the 5'-terminal base, and its genome organization deduced. The 5,677 nucleotide genome contains five large open reading frames (ORFs). The genes for the coat protein (1) and the putative viral RNA-dependent RNA polymerase were identified. The latter shows a striking degree of similarity to that of carnation mottle virus (CarMV). By comparison with corona- and retrovirus RNAs, it is proposed that a translational frameshift is involved in expression of the polymerase. An ORF encoding an Mr 49,797 protein (50K ORF) may be translated by in-frame readthrough of the coat protein stop codon. The coat protein, an overlapping 17K ORF, and a 3'6.7K ORF are likely to be expressed via subgenomic mRNAs.

Amino Acid Sequence↗
PubMed1994

Minute virus of mice infection modifies cellular transcription elongation.

Our previous observations indicated that upon infection with minute virus of mice (MVM), Ehrlich ascites cells lose a transcription elongation activity which is essential for the readthrough of the MVM attenuator. This was monitored by the ability of extracts from uninfected but not from infected cells to support readthrough of the P4 attenuator when added to partially purified transcription elongation complexes. We have investigated the nature of this change in transcription elongation following MVM infection. In this communication, we show that infection of Ehrlich ascites cells with MVM leads to a general shift in the length of nascent mRNA synthesized in isolated nuclei and separated by sucrose gradients. Furthermore, infection leads to attenuation of transcription of the cellular gene c-fos but not c-myc. We show biochemical evidence to support a model by which, following MVM infection, there is a functional reduction in the activity of a TFIIS-like general transcriptional elongation activity.

Animals↗
PubMed2005

Involvement of human release factors eRF3a and eRF3b in translation termination and regulation of the termination complex formation.

eRF3 is a GTPase associated with eRF1 in a complex that mediates translation termination in eukaryotes. In mammals, two genes encode two distinct forms of eRF3, eRF3a and eRF3b, which differ in their N-terminal domains. Both bind eRF1 and stimulate its release activity in vitro. However, whether both proteins can function as termination factors in vivo has not been determined. In this study, we used short interfering RNAs to examine the effect of eRF3a and eRF3b depletion on translation termination efficiency in human cells. By measuring the readthrough at a premature nonsense codon in a reporter mRNA, we found that eRF3a silencing induced an important increase in readthrough whereas eRF3b silencing had no significant effect. We also found that eRF3a depletion reduced the intracellular level of eRF1 protein by affecting its stability. In addition, we showed that eRF3b overexpression alleviated the effect of eRF3a silencing on readthrough and on eRF1 cellular levels. These results suggest that eRF3a is the major factor acting in translation termination in mammals and clearly demonstrate that eRF3b can substitute for eRF3a in this function. Finally, our data indicate that the expression level of eRF3a controls the formation of the termination complex by modulating eRF1 protein stability.

Amino Acid Sequence↗
PubMedDate not supplied

Position effect variegations and brain-specific silencing in transgenic mice overexpressing human acetylcholinesterase variants.

Position effect variegations as well as brain-specific silencing were observed in novel transgenic mouse pedigrees expressing human acetylcholinesterase (AChE) variants. Muscle AChE activities varied between 1.6- and 350-fold of control in these lines, one carrying insertion-inactivated InE6-AChE and two with 'readthrough' I4/E5 AChE, all under control of the ubiquitous CMV promoter. In contrast, brain AChE levels remained within a range of 1.5-fold over control, suggesting an upper limit of brain AChE which is compatible with life.

Acetylcholinesterase↗