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At least 487 records · Page 27Linked to original sources
PubMed1989

Characterization and nucleotide sequence of a novel gene fixW upstream of the fixABC operon in Rhizobium leguminosarum.

On the Rhizobium leguminosarum PRE sym plasmid, fixABC and a novel gene fixW were identified upstream of the regulatory gene nifA. The molecular masses of FixABC, 29, 44 and 50 kDa respectively, were estimated by polyacrylamide gel electrophoresis (PAGE) and of FixW, 25 kDa, by PAGE and nucleotide sequencing. Hybridization studies using bacteroid mRNA as a probe showed that fixABC is one operon which can be transcribed independently of fixW. Nucleotide sequencing revealed that both fixW and fixA are preceded by a nif consensus promoter. The fixA promoter partly overlaps the 3'-terminal coding region of fixW, indicating that readthrough from fixW into fixA is possible. Two open reading frames, ORF71 and ORF79, precede fixW and form one operon with fixW. ORF71 contains sequences homologous to the fixA promoter and 5'-terminal coding region. One more duplication of fixA sequences was detected, also located within the sym plasmid nif/fix clusters. One duplication of fixW sequences was found. No fixW homologue could be found in other nitrogen fixing organisms except in a number of R. leguminosarum strains.

Amino Acid Sequence↗
PubMed1986

The effect of the inverted repeat structure on the production of the cloned Bacillus amyloliquefaciens alpha-amylase.

An inverted repeat structure from Bacillus natto preceding the Bacillus subtilis alpha-amylase gene has been suggested to be responsible for the enhancement of alpha-amylase production [Yamazaki et al. (1983) J. Bacteriol. 156, 327-337]. A similar inverted repeat region has also been found upstream from the Bacillus amyloliquefaciens alpha-amylase gene and shown to function as a transcription termination signal of an upstream operon of 2.2 X 10(3) bases (2.2 kb) (Kallio et al., following paper in this journal). The removal of this DNA region reduced the level of alpha-amylase production by 70% and led to concomitant formation of a readthrough transcript arising from the promoter of the 2.2-kb operon. To test whether the readthrough transcript affected the alpha-amylase production, the promoter of the 2.2-kb operon was removed from plasmid constructions carrying either intact or deleted inverted repeat sequences. When cultures carrying the above plasmids were assayed for alpha-amylase activity, both constructions produced equal amounts of alpha-amylase. Thus, the inverted repeat structure preceding the alpha-amylase promoter does not, as such, enhance the alpha-amylase production. Instead, its presence prevents the inhibition of alpha-amylase expression caused by the readthrough transcripts from the upstream promoter.

Bacillus↗
PubMed2001

Targeting a nuclear anthranilate synthase alpha-subunit gene to the tobacco plastid genome results in enhanced tryptophan biosynthesis. Return of a gene to its pre-endosymbiotic origin.

Anthranilate synthase (AS), the control enzyme of the tryptophan (Trp) biosynthetic pathway, is encoded by nuclear genes, but is transported into the plastids. A tobacco (Nicotiana tabacum) cDNA (ASA2) encoding a feedback-insensitive tobacco AS alpha-subunit was transformed into two different sites of the tobacco plastid genome through site-specific insertion to obtain transplastomic plants with normal phenotype and fertility. A high and uniform level of ASA2 mRNA was observed in the transplastomic plants but not in the wild type. Although the plants with the transgene insertion at ndhF-trnL only expressed one size of the ASA2 mRNA, the plants with the transgene incorporated into the region between accD and open reading frame (ORF) 184 exhibited two species of mRNA, apparently due to readthrough. The transplastomic plants exhibited a higher level of AS alpha-subunit protein and AS enzyme activity that was less sensitive to Trp-feedback inhibition, leading to greatly increased free Trp levels in leaves and total Trp levels in seeds. Resistance to an AS inhibitor, 5-methyl-Trp, was found during seed germination and in suspension cultures of the transplastomic plants. The resistance to the selection agent spectinomycin and to 5-methyl-Trp was transmitted maternally. These results demonstrate the feasibility of modifying the biosynthetic pathways of important metabolites through transformation of the plastid genome by relocating a native gene from the nucleus to the plastid genome. Very high and uniform levels of gene expression can be observed in different lines, probably due to the identical insertion sites, in contrast to nuclear transformation where random insertions occur.

Anthranilate Synthase↗
PubMed2004

Leaky termination at premature stop codons antagonizes nonsense-mediated mRNA decay in S. cerevisiae.

The Nonsense-Mediated mRNA Decay (NMD) pathway mediates the rapid degradation of mRNAs that contain premature stop mutations in eukaryotic organisms. It was recently shown that mutations in three yeast genes that encode proteins involved in the NMD process, UPF1, UPF2, and UPF3, also reduce the efficiency of translation termination. In the current study, we compared the efficiency of translation termination in a upf1Delta strain and a [PSI(+)] strain using a collection of translation termination reporter constructs. The [PSI(+)] state is caused by a prion form of the polypeptide chain release factor eRF3 that limits its availability to participate in translation termination. In contrast, the mechanism by which Upf1p influences translation termination is poorly understood. The efficiency of translation termination is primarily determined by a tetranucleotide termination signal consisting of the stop codon and the first nucleotide immediately 3' of the stop codon. We found that the upf1Delta mutation, like the [PSI(+)] state, decreases the efficiency of translation termination over a broad range of tetranucleotide termination signals in a unique, context-dependent manner. These results suggest that Upf1p may associate with the termination complex prior to polypeptide chain release. We also found that the increase in readthrough observed in a [PSI(+)]/upf1Delta strain was larger than the readthrough observed in strains carrying either defect alone, indicating that the upf1Delta mutation and the [PSI(+)] state influence the termination process in distinct ways. Finally, our analysis revealed that the mRNA destabilization associated with NMD could be separated into two distinct forms that correlated with the extent the premature stop codon was suppressed. The minor component of NMD was a 25% decrease in mRNA levels observed when readthrough was >/=0.5%, while the major component was represented by a larger decrease in mRNA abundance that was observed only when readthrough was </=0.5%. This low threshold for the onset of the major component of NMD indicates that mRNA surveillance is an ongoing process that occurs throughout the lifetime of an mRNA.

Codon, Nonsense↗
PubMed2001

A novel stop codon readthrough mechanism produces functional Headcase protein in Drosophila trachea.

Translational regulation provides an efficient means to control the localization and production of proteins. The headcase (hdc) mRNA in Drosophila generates two overlapping proteins as a result of translational readthrough of an internal UAA stop codon. This readthrough event is necessary for the function of hdc as a branching inhibitor during tracheal development. By ectopic expression of different Hdc proteins in the trachea, we show that the long Hdc form alone, can function as a potent branching inhibitor whose activity is proportional to its amount. The suppression of termination in the hdc mRNA is not stop-codon dependent, suggesting that the readthrough does not involve codon specific suppressors. We have identified an 80 nucleotide sequence immediately downstream of the UAA, which is necessary and sufficient to confer termination readthrough in a heterologous mRNA. We present a novel mechanism of eukaryotic translational termination suppression that may regulate the amount of functional Hdc.

Animals↗
PubMed1994

The ompA 5' untranslated region impedes a major pathway for mRNA degradation in Escherichia coli.

The unusual longevity of the Escherichia coli ompA transcript is determined by its 5' untranslated region (UTR), which functions in vivo as an mRNA stabilizer. Here we show that this 5' UTR can prolong the lifetime in E. coli of a variety of heterologous mRNAs to which it is joined, either as a gene fusion or as an operon fusion. Statistical extrapolation suggests that it is quite likely that most E. coli mRNAs could be stabilized in this manner. We conclude that the ompA 5' UTR impedes a major pathway for mRNA degradation in E. coli and that stabilization by fusion to this UTR does not require translational readthrough of the heterologous mRNA segment by ribosomes that initiate translation at the ompA ribosome-binding site. Additional experiments indicate that the E. coli ribonuclease whose action is slowed by the ompA 5' UTR is not RNase III.

Bacterial Outer Membrane Proteins↗
PubMed2005

Selenocysteine insertion directed by the 3'-UTR SECIS element in Escherichia coli.

Co-translational insertion of selenocysteine (Sec) into proteins in response to UGA codons is directed by selenocysteine insertion sequence (SECIS) elements. In known bacterial selenoprotein genes, SECIS elements are located in the coding regions immediately downstream of UGA codons. Here, we report that a distant SECIS element can also function in Sec insertion in bacteria provided that it is spatially close to the UGA codon. We expressed a mammalian phospholipid hydroperoxide glutathione peroxidase in Escherichia coli from a construct in which a natural E.coli SECIS element was located in the 3'-untranslated region (3'-UTR) and adjacent to a sequence complementary to the region downstream of the Sec UGA codon. Although the major readthrough event at the UGA codon was insertion of tryptophan, Sec was also incorporated and its insertion was dependent on the functional SECIS element in the UTR, base-pairing potential of the SECIS flanking region and the Sec UGA codon. These data provide important implications into evolution of SECIS elements and development of a system for heterologous expression of selenoproteins and show that in addition to the primary sequence arrangement between UGA codons and SECIS elements, their proximity within the tertiary structure can support Sec insertion in bacteria.

3' Untranslated Regions↗
PubMed2001

Aminoglycoside-mediated suppression of nonsense mutations in late infantile neuronal ceroid lipofuscinosis.

The ability of aminoglycoside antibiotics to promote readthrough of eukaryotic stop codons has attracted interest in these drugs as potential therapeutic agents in human disorders caused by nonsense mutations. One disease for which such a therapeutic strategy may be viable is classical late infantile neuronal ceroid lipofuscinosis (LINCL), a fatal childhood neurodegenerative disorder with currently no effective treatment. Premature stop codon mutations in the gene CLN2 encoding the lysosomal tripeptidyl-peptidase 1 (TPP-I) are associated with disease in approximately half of children diagnosed with LINCL. The aim of this study was to examine the ability of the aminoglycoside gentamicin to restore TPP-I activity in LINCL cell lines. In one patient-derived cell line that was compound heterozygous for a commonly seen nonsense mutation, Arg208Stop and a different rare nonsense mutation, approximately 7% of normal levels of TPP-I were maximally restored with gentamicin treatment. In other cell lines from patients that were compound heterozygous for Arg208Stop and a splice junction mutation, approximately 0.5% of maximal activity was restored. These results suggest that pharmacological suppression of nonsense mutations by aminoglycosides or functionally similar pharmaceuticals may have therapeutic potential in LINCL.

Anti-Bacterial Agents↗
PubMed2004

Transactivator and structurally optimized inducible lentiviral vectors.

Lentiviral vectors offer well-recognized advantages as a gene delivery system both for the analysis of gene function and as a vehicle for gene therapy. In the present study optimized HIV-1-based vector systems that display efficient doxycycline (Dox)-dependent transgene expression in vitro and in vivo have been developed through the modification of factors that contribute to basal activity levels. Dissection of HIV-1 vectors harboring a tTA-dependent transgene expression cassette revealed several mechanisms that account for Dox-independent transgene expression, including those mediated by an internal CMV promoter, as well as a potential contribution from fusion proteins generated by translational readthrough. A precipitous reduction in basal activity levels was accomplished by separating the transactivator and the transgene cassettes into a binary vector system and by relocating the inducible promoter to the U3 region of the LTR. In addition, substituting the VP16 portion of tTA with the human p65 transactivating domain improved Dox-dependent transgene expression in a number of cell types. Optimizing HIV-1-based vectors culminated in a "toolbox" of vectors suitable for transgene delivery in vitro and in vivo, as conveyed by our ability to control the Dox-dependent differentiation of embryonic fibroblasts into muscle cells in vitro and transgene expression in rat brains.

Animals↗
PubMed2001

The 5'-untranslated region of the FMR1 message facilitates translation by internal ribosome entry.

Fragile X syndrome, the leading heritable form of mental impairment, is generally caused by large expansions of a CGG repeat in the promoter region of the FMR1 gene followed by transcriptional silencing. However, there is growing evidence that translation of the FMR1 message is also impaired, presumably because of the expanded CGG element in the 5'-untranslated region (5'-UTR) of the FMR1 message. To study the properties of the FMR1 5'-UTR, deletions were generated within a normal 5'-UTR with 16 CGG repeats for both monocistronic and dicistronic (luciferase) reporter constructs. Transient transfection experiments revealed a approximately 20-nucleotide region upstream of the CGG repeat element that functions as an internal ribosome entry site (IRES). The normal CGG element itself does not appear to influence the efficiency of IRES-mediated stimulation of downstream reporter activity (approximately 18-fold over controls). Additional controls indicate that the enhanced activity of the downstream reporter is not due to readthrough from the upstream cistron, nor is it due to translation of cryptic monocistronic transcripts. The role of the FMR1 IRES element is not known at present; however, by analogy to other IRES-containing mRNAs expressed in neurons, the FMR1 IRES element may help to promote translation in dendrites.

5' Untranslated Regions↗
PubMed1989

Rho-dependent transcription termination in the tyrT operon of Escherichia coli.

A 178-bp repeat sequence comprises the distal end of the tyrT operon and also encodes the signal for Rho-dependent transcription termination. It is shown here that Rho-dependent transcription termination is highly efficient for each of the individually cloned first and second repeats. The presence of either repeat results in a termination efficiency of 88% (12% readthrough). When an individual DNA repeat is shortened, starting from the promoter-proximal end, so that only 56 bp remain upstream from the termination site, Rho-dependent termination activity is reduced approximately three-fold (to give 38% readthrough) and with further shortening, so that only 15 bp remain upstream, Rho-dependent termination is reduced an additional 1.8-fold (to give 59% readthrough). Finally, it is shown that the presence or absence of the tyrT terminator upstream from the lambda tR1 terminator does not affect the efficiency of transcription at termination lambda tr1.

Base Sequence↗
PubMed1999

A sensitive assay of translational fidelity (readthrough and termination) in eukaryotic cells.

The process of translation termination in eukaryotes has been monitored by different types of assays, each with its own merits. We have developed an in vivo system where the reporter protein is secreted from the cells in culture thus enabling continuous monitoring of translation termination activity by simple sampling of the cell culture media. Using this system, cell cultures can be challenged with various stimuli during growth and the cellular responses on the translational level can be investigated in vivo as well as in vitro. Sampling is rapid, easy, and non-destructive to the cells, which enables measurement of translational fidelity in real time during time-course experiments. In particular with this system it is possible to assess very low levels of stop codon suppression. The reporter enzyme, secreted alkaline phosphatase (SEAP), becomes tagged with the S-peptide when there is readthrough of a stop codon placed between the C-terminus of the SEAP and the S-peptide. The tagged SEAP is bound to a matrix and the bound SEAP activity is measured versus total SEAP activity in the medium as a reference. With this assay we have confirmed that eRF1 acts as an antisuppressor in cells transfected with a cognate suppressor tRNA as well as in control cells, where a small but significant level of readthrough (suppression) could be detected. We have also characterized suppression of the three stop codons individually, and especially UGA is prone for wobbling.

Alkaline Phosphatase↗
PubMed1994

Identification of a locus control region in the immunoglobulin heavy-chain locus that deregulates c-myc expression in plasmacytoma and Burkitt's lymphoma cells.

In murine plasmacytoma and human Burkitt's lymphoma cells, one allele of c-myc is translocated into one of the immunoglobulin loci, resulting in a characteristic pattern of deregulated c-myc transcription. Translocation events between c-myc and the IgH locus segregate c-myc and the IgH intron enhancer to different reciprocal products in all plasmacytomas and in most Burkitt's lymphoma cells, suggesting that an additional element(s) capable of affecting c-myc expression over a large and variable distance must exist in the IgH locus. The region 3' of the IgH C alpha gene contains four tissue-specific and cell stage-specific DNase I hypersensitive sites (HSs), two of which map to the late B cell-specific 3' C alpha enhancer. We report here that DNA sequences comprising the two other 3' C alpha HSs contain potential protein-binding motifs for trans-activators commonly associated with immunoglobulin enhancers and that these sites can function as cell stage-specific enhancers in transient B cell assays. A DNA fragment containing all four HSs (HS1234) synergistically activates c-myc transcription in plasmacytoma and Burkitt's lymphoma cells in transient assays and induces high-level transcription, a promoter shift from P2 to P1, and an increase in readthrough transcription in stable transfections. Furthermore, plasmacytoma clones stably transfected with a HS1234-linked c-myc construct express c-myc in a position-independent, copy number-dependent manner. These results suggest that HS1234 may function as a locus control region (LCR), deregulating c-myc expression in t(15;12) plasmacytomas, as well as potentially contributing to aspects of normal IgH chain expression.

Animals↗
PubMed1998

Peroxisomal and mitochondrial carnitine acetyltransferase isozymes of the n-alkane-assimilating yeast, Candida tropicalis, occurred by alternative initiation of translation from the transcripts of a single gene.

Carnitine acetyltransferase (CAT; EC 2.3.1.7) is localized in two subcellular organelles, peroxisomes and mitochondria, in an n-alkane-assimilating yeast, Candida tropicalis. The isozymes are synthesized from the first and second ATG codon of the open reading frame of one gene, CtCAT. Primer extension analysis and RNase protection assay (RPA) revealed that multiple transcription initiation sites were found upstream of the first ATG codon. 5' ends could not be detected between the first and second ATG codons. These results suggested that the peroxisomal CAT of C. tropicalis, initiating at the second AUG codon of the transcripts, was synthesized by a translational readthrough of the first AUG codon of the open reading frame. When CtCAT was introduced into the other yeast, Saccharomyces cerevisiae, 5' ends of transcripts and the protein products were similar to those observed in C. tropicalis. This suggested that the transcripts harbored sufficient information to bring about alternative initiation of translation in both yeasts. Using S. cerevisiae as the host cell, introduction of mutations into the sequence near the first AUG codon or a deletion in the region between the first and second AUG codons resulted in an increased ratio of translation from the first AUG codon, although initiation sites of transcription did not change. Moreover, replacing the 5' leader sequence to that of C. tropicalis isocitrate lyase promoter (UPR-ICL) eliminated the product initiating at the second AUG codon. The transcript from these cells was shorter than those detected from the native CtCAT-harboring cells. From these results, it was strongly suggested that peroxisomal and mitochondrial CAT isozymes occurred by the alternative initiation of translation mainly dependent on the structure and sequence context of the region from the 5' end to the second AUG codon, and not the insufficient length of the 5' leader.

Amino Acid Sequence↗
PubMed2005

A novel peroxisome proliferator-activated receptor gamma isoform with dominant negative activity generated by alternative splicing.

We examined the peroxisome proliferator-activated receptor gamma (PPARG) locus in an attempt to identify expressed sequence tags and/or conserved non-coding sequences in the intron sequences containing open reading frames and potentially able to encode new proteins. We identified a new PPARG transcript, defined gammaORF4, which harbors a readthrough in intron 4. The expected translated protein lacks the ligand-binding domain encoded by exons 5 and 6. We identified the transcript in human tumor cell lines and tissues, synthesized the cDNA, and cloned it in expression vectors. Using transient transfections, we found that gammaORF4 cDNA is translated into a predominantly nuclear protein that does not transactivate a reporter gene. Moreover, the isoform is dominant negative versus PPARgamma. Interestingly, gammaORF4 was expressed in vivo in a series of sporadic colorectal cancers. In some cases, it was expressed, albeit at lower levels, also in the mucosa adjacent to the tumors, suggesting that it may be related to tumorigenesis. A tumorigenic effect of gammaORF4 is in line with our finding that gammaORF4 has not only lost the capacity to restrain cell growth but has acquired the potential to stimulate it. In conclusion, this study demonstrates that gammaORF4 is expressed in vivo, that it has lost some PPARgamma properties, and that it affects PPARgamma functioning. The ability to counteract PPARgamma suggests that gammaORF4 plays a role in the pathogenesis of colorectal cancers.

Alternative Splicing↗
PubMed1997

Co-packaging of non-vector RNAs generates replication-defective retroviral vector particles: a novel approach for blocking retrovirus replication.

A Moloney murine leukemia virus (MoMuLV)-derived packaging retroviral vector, pUCMoTN-PR3, was previously developed in which the packaging (psi) signal was cloned within the 5'-long terminal repeat (LTR) U3-r and U5 sequences. The MoTN-PR3 vector particles released from a transfected packaging cell line contain RNAs with r-psi-U5 sequences at the 5'-end and U3-r sequences at the 3'-end. Upon infection, these vector particles can efficiently transduce the neomycin phosphotransferase (neo) gene to the target cells. The structure of the proviral DNA synthesized in these cells was shown to contain modified 5'- and 3'-LTRs with U3-r-psi-U5 sequences, indicating that this vector can undergo reverse transcription and integration. Analysis of psi signal-containing RNAs revealed that in addition to vector RNA transcribed from the MoMuLV 5'-LTR promoter, readthrough neo RNA transcribed from the internal herpes simplex virus (HSV) thymidine kinase (tk) promoter and cellular RNAs transcribed from the MoMuLV 3'-LTR promoter are produced. Of these, the downstream cellular RNAs are also packaged within the vector particles. These vector particles containing the vector and non-vector RNAs carrying the MoMuLV psi signal are non-infectious. It is proposed that intracellular expression of packageable non-viral RNAs may represent an effective strategy for inhibiting animal and plant virus replication.

3T3 Cells↗
PubMed1991

Sequence of a cDNA carrying the glycoprotein gene and part of the matrix protein M2 gene of viral haemorrhagic scepticaemia virus, a fish rhabdovirus.

A cDNA clone encoding for the glycoprotein of the viral haemorrhagic scepticaemia virus, a fish rhabdovirus, has been sequenced. The cDNA was 2035 bp long and contained two open reading frames (ORF). A 1523 bp ORF corresponded to the glycoprotein and was adjacent, on its 5' side, to an incomplete 372 bp ORF. Although the protein encoded by this ORF displayed no similarity with other rhabdovirus proteins, it was supposed that the cDNA had been reverse-transcribed from a readthrough mRNA encoding successively for the M2 and the G proteins.

Amino Acid Sequence↗
PubMed2004

Translocation of Escherichia coli RNA polymerase against a protein roadblock in vivo highlights a passive sliding mechanism for transcript elongation.

Current models for transcription elongation infer that RNA polymerase (RNAP) moves along the template by a passive sliding mechanism that takes advantage of random lateral oscillations in which single basepair sliding movements interconvert the elongation complex between pre- and post-translocated states. Such passive translocational equilibrium was tested in vivo by a systematic change in the templated NTP that is to be incorporated by RNAP, which is temporarily roadblocked by the lac repressor. Our results show that, under these conditions that hinder the forward movement of the polymerase, the elongation complex is able to extend its RNA chain one nucleotide further when the incoming NTP is a kinetically favoured substrate (i.e. low K(m)). The addition of an extra nucleotide destabilizes the repressor-operator roadblock leading to an increase in transcriptional readthrough. Similar results are obtained when the incoming NTPs are less kinetically favoured substrates (i.e. high K(m)s) by specifically increasing their intracellular concentrations. Altogether, these in vivo data are consistent with a passive sliding model in which RNAP forward translocation is favoured by NTP binding. They also suggest that fluctuations in the intracellular NTP pools may play a key role in gene regulation at the transcript elongation level.

Bacterial Proteins↗