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PubMed1989

Nucleotide sequence of potato leafroll luteovirus RNA.

A sequence of 5987 nucleotides is reported for the RNA of potato leafroll luteovirus (PLRV). The sequence contains six large open reading frames, and non-coding regions of 174 nucleotides at the 5' end, 141 nucleotides at the 3' end and 197 nucleotides between two large blocks of coding sequences. The 5' coding region encodes two polypeptides of 28,000 (28K) and 70K which overlap in different reading frames and circumstantial evidence suggests that the third open reading frame in the 5' block is translated by frameshift readthrough near the end of the 70K polypeptide to give a 118K polypeptide. The C-terminal part of the 118K protein contains the consensus sequence for RNA-dependent RNA polymerases. In vitro translation of PLRV RNA resulted in the synthesis mainly of 28K and 70K polypeptides and the largest product made was about 125K; these sizes are similar to those predicted for the translation products of the 5' block of coding sequence. The 3' block of coding sequence codes for three polypeptides: a 23K coat protein, a 17K polypeptide which is encoded in a different frame, and a 53K polypeptide which immediately follows the coat protein coding sequence, and is in the same reading frame. Circumstantial evidence suggests that the 53K polypeptide is translated by readthrough of the amber termination codon of the coat protein gene. The amino acid sequences encoded by the 3' block of coding sequence show many similarities with analogous polypeptides translated from the nucleotide sequences of RNA of barley yellow dwarf virus, PAV strain (BYDV) and, in particular, beet western yellows virus (BWYV). The 118K polypeptide has some similarities with the putative polymerase of southern bean mosaic virus and much more extensive similarities with the corresponding BWYV polypeptide but almost none with that of BYDV. In contrast, the amino acid sequence of the 28K polypeptide is not like that of proteins of the other luteoviruses or of viruses in other groups. The nucleotide sequences reported will appear in the EMBL, GenBank and DDBJ databases under the accession number X14600.

Amino Acid Sequence↗
PubMed2001

Display of passenger proteins on the surface of Escherichia coli K-12 by the enterohemorrhagic E. coli intimin EaeA.

Intimins are members of a family of bacterial adhesins from pathogenic Escherichia coli which specifically interact with diverse eukaryotic cell surface receptors. The EaeA intimin from enterohemorrhagic E. coli O157:H7 contains an N-terminal transporter domain, which resides in the bacterial outer membrane and promotes the translocation of four C-terminally attached passenger domains across the bacterial cell envelope. We investigated whether truncated EaeA intimin lacking two carboxy-terminal domains could be used as a translocator for heterologous passenger proteins. We found that a variant of the trypsin inhibitor Ecballium elaterium trypsin inhibitor II (EETI-II), interleukin 4, and the Bence-Jones protein REI(v) were displayed on the surface of E. coli K-12 via fusion to truncated intimin. Fusion protein net accumulation in the outer membrane could be regulated over a broad range by varying the cellular amount of suppressor tRNA that is necessary for translational readthrough at an amber codon residing within the truncated eaeA gene. Intimin-mediated adhesion of the bacterial cells to eukaryotic target cells could be mimicked by surface display of a short fibrinogen receptor binding peptide containing an arginine-glycine-aspartic acid sequence motif, which promoted binding of E. coli K-12 to human platelets. Cells displaying a particular epitope sequence fused to truncated intimin could be enriched 200,000-fold by immunofluorescence staining and fluorescence-activated cell sorting in three sorting rounds. These results demonstrate that truncated intimin can be used as an anchor protein that mediates the translocation of various passenger proteins through the cytoplasmic and outer membranes of E. coli and their exposure on the cell surface. Intimin display may prove a useful tool for future protein translocation studies with interesting biological and biotechnological ramifications.

Adhesins, Bacterial↗
PubMed2006

Readthrough strategies for stop codons in Duchenne muscular dystrophy.

Duchenne muscular dystrophy is due to mutations of the dystrophin gene. These are large deletions or duplications in 80% of cases, while premature stop codons (nonsense point mutations) account for 7% of cases. This subgroup of patients may take advantage of the properties of the antibiotic gentamicin to suppress stop codons (readthrough). The efficiency of the readthrough varies inversely to the efficiency of a stop codon and is also affected by the different components of the drug. Following gentamicin treatment of mdx mice, dystrophin was re-expressed up to 20% of normal level, albeit with variability among animals. Human trials with gentamicin have so far obtained doubtful results. PTC124 belongs to a new class of small molecules that mimics at lower concentrations the readthrough activity of gentamicin. The administration of PTC124 resulted in the production of full-length and functionally active dystrophin both in vitro and in mdx mice. A Phase II clinical trial is now in course and will be terminated at the end of 2006.

Aminoglycosides↗
PubMed2006

Translational readthrough induction of pathogenic nonsense mutations.

The treatment of genetic disorders is one of the biggest challenges lying ahead of modern medicine. While major advancements have been made in gene therapy, it is still far from achieving clinical success. However, other potential methods for treating single gene related diseases have also emerged recently. One such approach is the suppression of pathogenic nonsense mutations through inducing translational readthrough of the in-frame premature stop mutation. Aminoglycosides were the first drugs that gave promising results in this respect. This report provides a brief overview on the past, present and potential future of this pharmacogenetic approach.

Aminoglycosides↗
PubMed2026

Duchenne muscular dystrophy: from gene to gene-ius therapies.

Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional dystrophin, leading to progressive muscle degeneration, loss of ambulation, respiratory failure, cardiomyopathy, and premature mortality. Despite advances in multidisciplinary supportive care, DMD remains an incurable disease associated with substantial physical, psychosocial, and economic burdens. The monogenic nature of DMD and its well-defined molecular pathogenesis have made it a prime target for the development of precision therapies aimed at restoring dystrophin expression or modifying disease progression. This review provides an overview of the genetic and molecular mechanisms underlying DMD, summarizes its clinical manifestations and natural history, and discusses current standards of care. It further examines recent advances in disease-modifying therapeutic strategies, including exon-skipping antisense oligonucleotides, nonsense mutation readthrough agents, adeno-associated virus (AAV)-mediated micro-dystrophin gene replacement, and emerging genome-editing technologies such as CRISPR/Cas9. The review also highlights the limitations of existing treatments, including mutation specificity, variable efficacy, immune-related challenges, and uncertainties regarding long-term durability and safety. Finally, it considers future directions in therapeutic development, emphasizing the need for combination approaches, improved delivery systems, and next-generation gene-editing platforms to achieve more effective and lasting clinical outcomes. Collectively, these advances represent a paradigm shift in DMD management and offer renewed hope for improving survival and quality of life for affected individuals.

Humans↗
PubMed1993

A new cloning vector and expression strategy for genes encoding proteins toxic to Escherichia coli.

Here, we describe a modification of a plasmid, pT7-7 [Tabor and Richardson, Proc. Natl. Acad. Sci. USA 262 (1985) 1074-1078], that allows expression of inserted genes from the phage T7 RNA polymerase promoter. The modification is designed to suppress readthrough transcription from cryptic promoters and start points on the plasmid, in order to reduce expression in the absence of T7 RNA polymerase and thus improve the vector for use in the expression of highly toxic gene products. This vector (pT7SC) was used to stably clone the POL3 gene (encoding DNA polymerase delta) of Saccharomyces cerevisiae, which destabilizes all other cloning and expression vectors tested. Previously described expression strategies proved ineffective in overexpressing the POL3 gene. A new strategy was developed which relies on induction by infection with mutant T7 phage. This system efficiently overproduced the POL3 gene product.

Bacteriophage T7↗
PubMed2004

Immediate early genes expressed in chlorovirus infections.

Twenty-three chlorovirus genes expressed in host cells as early as 5-10 min postinfection (p.i.), or immediate early, were isolated and characterized. Some showed significant homology with those for transcriptional factors and mRNA-processing proteins including TFIIB, helicases, mRNA capping enzyme, nucleolin, and bean transcription factor. Others code for (i) factors influencing translation such as aminoacyl tRNA synthetases and ribosomal protein, and (ii) unknown proteins. Enzymes involved in polysaccharide synthesis were also found. All transcripts of these genes had a poly(A) tail, which decreased in size after 20 min p.i., possibly caused by the shortening by an exonuclease. Often, due to readthrough either from an upstream ORF or into a downstream ORF, a few extra transcripts for each gene appeared after 40 min p.i., suggesting a change in promoter selection and termination accuracy at this point. A typical TATA-box and a common element 5'-ATGACAA were in the promoter region of almost all of the immediate early genes, which may be recognized by host RNA polymerase and transcription factors.

Animals↗
PubMed2003

Variations in transcription termination signals of human respiratory syncytial virus clinical isolates affect gene expression.

Human respiratory syncytial virus (HRSV) has a single-stranded, negative-sense RNA genome with 10 genes encoding 11 proteins. Sequences at the beginning of the HRSV genes are highly conserved; however, the gene end sequences vary around a semiconserved consensus sequence, and the nontranscribed intergenic regions vary in both length and sequence. The regions at the junctions between HRSV genes (the gene end sequence of an upstream gene, intergenic region, and the gene start sequence of a downstream gene) contain elements required for efficient termination of the upstream gene and transcription of the downstream gene. Previous studies have examined variation in the HRSV coding sequences, but none have systematically analyzed the noncoding transcriptional control regions for variability. We determined the gene start and gene end sequences of each of the 10 HRSV genes from 14 clinical isolates for variations from the sequence of the prototype A2 strain. No changes were found in any of the gene start sequences. Eight of the 10 gene end sequences, however, contained variations. Several of these, a U(4)-tract instead of a U(6)- or U(5)-tract at the M and SH gene ends, respectively, (U(4)A) and an A-to-G change at position four in the G gene end (A4G), were predicted to affect termination and were examined for their effects on transcription. The changes were found to inhibit transcriptional termination, resulting in increased polycistronic readthrough and correspondingly reduced initiation of the downstream monocistronic mRNA. Viruses with the A4G variant G gene end sequence produced less F protein than those with A2-like G gene end sequences. Examination of additional G gene end sequences available in GenBank revealed that the observed A4G variation was restricted to one phylogenetic lineage of HRSV. All viruses examined within this lineage possessed this variant G gene end sequence. The data presented show that the gene end sequences of naturally occurring HRSV clinical isolates vary from those of the prototypic A2 strain and that certain of these changes inhibit efficient transcriptional termination and downstream gene expression.

Gene Expression Regulation↗
PubMed1996

Activator-dependent regulation of transcriptional pausing on nucleosomal templates.

Promoter-proximal pausing during transcriptional elongation is an important way of regulating many diverse genes, including human c-myc and c-fos, some HIV genes, and the Drosophila heat shock loci. To characterize the mechanisms that regulate pausing, we have established an in vitro system using the human hsp7O gene. We demonstrate that nucleosome formation increases by >100-fold the duration of a transcriptional pause on the human hsp7O gene in vitro at the same location as pausing is observed in vivo. Readthrough of this pause is increased by an activator that contains the human heat shock factor 1 (HSF1) transcriptional activation domains. Maximal effect of the activator requires that the system be supplemented with fractions that have hSWI/SNF activity, which has been shown previously to alter nucleosome structure. No significant readthrough is observed in the absence of activator, and neither the activator nor the hSWI/SNF fraction affected elongation on naked DNA; therefore, these results suggest that an activator can cause increased readthrough of promoter-proximal pausing by decreasing the inhibitory effect of nucleosomes on transcriptional elongation.

Base Sequence↗
PubMed2002

Lack of pseudouridine 38/39 in the anticodon arm of yeast cytoplasmic tRNA decreases in vivo recoding efficiency.

Many different modified nucleotides are found in naturally occurring tRNA, especially in the anticodon region. Their importance for the efficiency of the translational process begins to be well documented. Here we have analyzed the in vivo effect of deleting genes coding for yeast tRNA-modifying enzymes, namely Pus1p, Pus3p, Pus4p, or Trm4p, on termination readthrough and +1 frameshift events. To this end, we have transformed each of the yeast deletion strains with a lacZ-luc dual-reporter vector harboring selected programmed recoding sites. We have found that only deletion of the PUS3 gene, encoding the enzyme that introduces pseudouridines at position 38 or 39 in tRNA, has an effect on the efficiency of the translation process. In this mutant, we have observed a reduced readthrough efficiency of each stop codon by natural nonsense suppressor tRNAs. This effect is solely due to the absence of pseudouridine 38 or 39 in tRNA because the inactive mutant protein Pus3[D151A]p did not restore the level of natural readthrough. Our results also show that absence of pseudouridine 39 in the slippery tRNA(UAG)(Leu) reduces +1 frameshift efficiency. Therefore, the presence of pseudouridine 38 or 39 in the tRNA anticodon arm enhances misreading of certain codons by natural nonsense tRNAs as well as promotes frameshifting on slippery sequences in yeast.

Anticodon↗
PubMed1998

Regulation of the Enterococcus faecalis pAD1-related sex pheromone response: analyses of traD expression and its role in controlling conjugation functions.

The Enterococcus faecalis haemolysin plasmid pAD1 (60 kb) confers a conjugative mating response to an octapeptide sex pheromone (cAD1) secreted by plasmid-free strains. The response involves two plasmid-borne regulatory determinants: traE1, whose product positively regulates all or most conjugation-related structural genes; and traA, whose product negatively regulates traE1 by controlling transcriptional readthrough of an upstream termination site (TTS1/TTS2). TraA binds to the promoter region of iad, which encodes a pheromone-inhibitor peptide, iAD1; and TTS1/TTS2 tightly terminates transcription arriving from this promoter during the uninduced state. A determinant, traD, appearing to encode a small peptide (23 amino acids), is located just downstream of iad and is in the opposite orientation. Transcripts of traD were identified and found to be present at a relatively high level in cells not expressing conjugation functions; the amount of RNA was greatly reduced, however, upon induction of the pheromone response. The decrease in traD RNA was not a consequence of the induced activity of TraE1, as it also occurred in a traE1 insertion mutant. A mutation in traD that would eliminate translation but that did not affect transcription had no apparent effect on the cell phenotype, indicating that RNA was likely to be the functional product. This was consistent with our finding that synthesis of traD RNA containing the translational defect was able to complement, in trans, a temperature-sensitive traD mutation. Thus, transcription of the traD determinant is significantly involved in downregulation of the pAD1 pheromone response.

Amino Acid Sequence↗
PubMed1999

Cloning of a long HIV-1 readthrough transcript and detection of an increased level of early growth response protein-1 (Egr-1) mRNA in chronically infected U937 cells.

To identify the pathways involved in HIV-1 modification of cellular gene expression, chronically infected U937 cells were screened by mRNA differential display. A chimeric transcript consisting of the 3' end of the LTR of a HIV-1 provirus, followed by 3.7 kb of cellular RNA was identified suggesting that long readthrough transcription might be one of the mechanisms by which gene expression could be modified in individual infected cells. Such a phenomenon may also be the first step towards the potential transduction of cellular sequences. Furthermore, the mRNA encoding for the transcription factor Egr-1 was detected as an over-represented transcript in infected cells. Northern blot analysis confirmed the increase of Egr-1 mRNA content in both HIV-1 infected promonocytic U937 cells and T cell lines such as Jurkat and CEM. Interestingly a similar increase of Egr-1 mRNA has previously been reported to occur in HTLV-1 and HTLV-2 infected T cell lines. Despite the consistent increase in the level of Egr-1 mRNA, the amount of the encoded protein did not appear to be modified in HIV-1 infected cells, suggesting an increased turn over of the protein in chronically infected cells.

Cloning, Molecular↗
PubMed1998

Functional and genetic analysis of regulatory regions of coliphage H-19B: location of shiga-like toxin and lysis genes suggest a role for phage functions in toxin release.

Analysis of the DNA sequence of a 17 kb region of the coli lambdoid phage H-19B genome located the genes encoding shiga-like toxin I (Stx-I) downstream of the gene encoding the analogue of the phage lambda Q transcription activator with its site of action, qut at the associated pR' late promoter, and upstream of the analogues of lambda genes encoding lysis functions. Functional studies, including measurement of the effect of H-19B Q action on levels of Stx expressed from an H-19B prophage, show that the H-19B Q acts as a transcription activator with its associated pR'(qut) by promoting readthrough of transcription terminators. Another toxin-producing phage, 933W, has the identical Q gene and pR'(qut) upstream of the stx-II genes. The H-19B Q also activates Stx-II expression from a 933W prophage. An ORF in H-19B corresponding to the holin lysis genes of other lambdoid phages differs by having only one instead of the usual two closely spaced translation initiation signals that are thought to contribute to the time of lysis. These observations suggest that stx-I expression can be enhanced by transcription from pR' as well as a model for toxin release through cell lysis mediated by action of phage-encoded lysis functions. Functional studies show that open reading frames (ORFs) and sites in H-19B that resemble components of the N transcription antitermination systems controlling early operons of other lambdoid phages similarly promote antitermination. However, this N-like system differs significantly from those of other lambdoid phages.

Amino Acid Sequence↗
PubMed1989

Alteration of hsp82 gene expression by the gypsy transposon and suppressor genes in Drosophila melanogaster.

Several mutations in Drosophila result from insertion of the gypsy retrotransposon. Gypsy insertion mutagenesis and its modulation by allele-specific modifier genes were investigated by inserting gypsy or fragments of it into the intron of the Drosophila hsp82 heat shock gene. With gypsy in the parallel orientation, nearly all transcripts in transfected cells and transformed pupae were truncated in the 5' long terminal repeat (LTR). Truncation also occurred in or near the 3' LTR. The 5' LTR polyadenylation signal was strongly potentiated by a downstream 326-bp internal gypsy segment in either orientation. Anti-parallel gypsy reduced the amount of normal transcript to a much smaller extent, and a low level of truncation occurred within gypsy. No evidence was found for effects of the gypsy insertions on the hsp82 promoter. Mutations in the allelespecific modifier genes su(f) and su(w alpha) had effects on the amounts of readthrough transcripts consistent with their genetic behavior, whereas the effects of mutations in su(Hw) were only partly in accord with genetic expectations.

Animals↗
PubMed1987

Novel amber suppressor tRNAs of mammalian origin.

Two amber suppressor tRNAs have been isolated from calf liver. They are different from previously identified naturally occurring amber suppressors of eukaryotes in so far as they are neither tRNATyr nor tRNAGln. They are leucine iso-acceptors and their nucleotide sequence indicates that they harbour a CAA and a CAG anticodon respectively. Both species are functional as amber suppressors as demonstrated by readthrough of the amber codon which terminates the 126 kd protein gene of tobacco mosaic virus RNA. The results bring new information in the discussion of codon-anticodon recognition and regulation of termination in eukaryotic protein synthesis.

Animals↗
PubMed1980

Cloning of an EcoRI-generated fragment of the leucine operon of Salmonella typhimurium.

Recombinant plasmids carrying part of the leucine operon of Salmonella typhimurium were isolated following transformation of an Escherichia coli leucine auxotroph to prototrophy with a ligated mixture of EcoRI-treated Salmonella DNA and plasmid pSC101 DNA. Plasmids pCV11 and pCV13, containing a 3.4-10(6) dalton DNA fragment ligated to the vector, had the leu operon oriented in opposite directions. The orientation of the leu operon relative to plasmid genes was determined. The 3.4-10(6) dalton fragment was ligated in to the EcoRI site of plasmid pMB9 yielding plasmids pCV12 (orientation as in pCV11) and pCV14 (orientation as in pCV13). The results of enzyme assays and complementation tests indicated that these plasmids carry functional leuA, leuB, and leuC genes but not a functional leuD gene. Furthermore, the following results indicated that they have a functional leu control region and promoter. Expression of plasmid leu genes was markedly enhanced under conditions of leucine limitation whereas introduction of a leu promoter mutation into the operon oriented in either direction with respect to plasmid genes had a strong negative effect upon leu operon expression. Transcriptional readthrough from plasmid promoters, if it occurs at all, must be small in comparison with transcription initiated at the leu promoter. RNA was isolated from leucine auxotrophs grown under conditions of repression and derepression and from prototrophic strains derepressed for the leucine operon as a result of mutations in leuO, leuS, and flrB. The rate of synthesis of leu mRNA, measured by hybridization to plasmid pCV12 DNA, was proportional in each case to leu enzyme levels.

Cloning, Molecular↗
PubMed1986

Construction and characterization of pBR322-derived plasmids with deletions of the RNA I region.

A region upstream from the origin of replication in ColE1-type plasmids has been shown to be necessary for replication. Two RNA transcripts are produced from this area, RNA II, which yields the primer for DNA polymerase initiation at the origin and RNA I, which is complementary to the 5' end of RNA II and acts to inhibit primer formation. We have constructed plasmids which do not possess the nucleotide sequence for RNA I, or the normal 5' terminus and promoter of RNA II. The RNA II analog, in these plasmids, is believed to be synthesized by the readthrough transcription of the upstream trimethoprim-resistant dihydrofolate reductase (DHFR) gene at a level comparable to that produced by the tryptophan promoter. These plasmids have a copy number of about tenfold higher than that of pBR322 during logarithmic growth and are compatible with other ColE1-type plasmids. These plasmids are stably maintained in several strains when selective pressure is present and the plasmids are stably maintained during exponential growth in W3110 strains without selective pressure. In all strains examined, the dimeric form of the plasmid was lost from cells much more rapidly than those containing the monomeric form.

Base Sequence↗
PubMed1998

Polyadenylation of vesicular stomatitis virus mRNA dictates efficient transcription termination at the intercistronic gene junctions.

The intercistronic gene junctions of vesicular stomatitis virus (VSV) contain conserved sequence elements that are important for polyadenylation and transcription termination of upstream transcript as well as reinitiation of transcription of downstream transcript. To examine the role of the putative polyadenylation signal 3'AUACU(7)5' at the gene junctions in polyadenylation and transcription termination, we constructed plasmids encoding antigenomic minireplicons containing one or two transcription units. In plasmid-transfected cells, analyses of the bicistronic minireplicon containing the wild-type or mutant intercistronic gene junctions for the ability to direct synthesis of polyadenylated upstream, downstream, and readthrough mRNAs showed that the AUACU(7) sequence element is required for polyadenylation of VSV mRNA. Deletion of AUAC or U(7) resulted in templates that did not support polyadenylation of upstream mRNA. Interestingly, we found that the loss of polyadenylation function led to antitermination of the upstream transcript and resulted in a readthrough transcript that contained the upstream and downstream mRNA sequences. Mutations that blocked polyadenylation also blocked transcription termination and generated mostly readthrough transcript. Reverse transcription-PCR of readthrough transcripts and subsequent nucleotide sequencing of the amplified product revealed no extra adenosine residues at the junction of the readthrough transcript. These results indicate that polyadenylation is required for transcription termination of VSV mRNA. The intergenic dinucleotide GA did not appear to be necessary for transcription termination. Furthermore, we found that insertion of the polyadenylation signal sequence AUACU(7) alone was sufficient to direct polyadenylation and efficient transcription termination at the inserted site. Taken together, the data presented here support the conclusion that polyadenylation is the major determinant of transcription termination at the intercistronic gene junctions of VSV.

Adenosine↗