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.

SourceResearch connectionFrom yearThrough year

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

623 records · Page 35Linked to original sources

PubMed2001

Modified testicular expression of stress-associated "readthrough" acetylcholinesterase predicts male infertility.

Male infertility is often attributed to stress. However, the protein or proteins that mediate stress-related infertility are not yet known. Overexpression of the "readthrough" variant of acetylcholinesterase (AChE-R) is involved in the cellular stress response in a variety of mammalian tissues. Here, we report testicular overexpression of AChE-R in heads, but not tails, of postmeiotic spermatozoa from mice subjected to a transient psychological stress compared with age-matched control mice. Transgenic mice overexpressing AChE-R displayed reduced sperm counts, decreased seminal gland weight, and impaired sperm motility compared with age-matched nontransgenic controls. AChE-R was prominent in meiotic phase spermatocytes and in tails, but not heads, of testicular spermatozoa from AChE-R transgenic mice. Head-localized AChE-R was characteristic of human sperm from fertile donors. In contrast, sperm head AChE-R staining was conspicuously reduced in samples from human couples for whom the cause of infertility could not be determined, similar to the pattern found in transgenic mice. These findings indicate AChE-R involvement in impaired sperm quality, which suggests that it is a molecular marker for stress-related infertility.

Acetylcholinesterase↗

PubMed2001

The readthrough region of Potato mop-top virus (PMTV) coat protein encoding RNA, the second largest RNA of PMTV genome, undergoes structural changes in naturally infected and experimentally inoculated plants.

Molecular data on Potato mop-top virus (PMTV), genus Pomovirus, is currently mostly based on analysis of two Scottish isolates, PMTV-S and PMTV-T. Here we report the complete sequence of "the coat protein (CP) encoding RNA" of an isolate of PMTV obtained from the field in Sweden. Our data show that this RNA (3134 nt) is the second largest of the three RNA species in the tripartite PMTV genome, and it should, therefore, be referred to as RNA 2. This nomenclature is consistent with other pomoviruses. The sequence of the readthrough domain (RT) of RNA 2 was determined also in two additional field isolates of PMTV from Finland and Denmark. All three isolates contained a novel, 109 nucleotides long sequence at the 3'-end of the RT, which has not been found in PMTV-S and PMTV-T. Hence, our data suggest that the RNA 2 sequences previously described for the isolates PMTV-T and PMTV-S may represent deletion derivatives. The C-proximal half of RT contained many amino acid (aa) differences among the isolates, in contrast to only few aa differences in the N-proximal part of RT. Deletion variants of RNA 2 were generated from the Nordic isolates in potato tubers infected in the field, and in the mechanically inoculated test plants. All deletions started within a short region (18 nt) and removed 558-940 nt from the 3'-end of RT region. This study for the first time describes the full-length sequence of the "CP-encoding RNA" (RNA2) of PMTV, and reveals considerable aa variability and occurrence of deletion variants of RT in the field isolates of PMTV.

Amino Acid Sequence↗

PubMed1996

The retrotransposon Tf1 assembles virus-like particles that contain excess Gag relative to integrase because of a regulated degradation process.

The retrotransposon Tf1, isolated from Schizosaccharomyces pombe, contains a single open reading frame with sequences encoding Gag, protease, reverse transcriptase, and integrase (IN). Tf1 has previously been shown to possess significant transposition activity. Although Tf1 proteins do assemble into virus-like particles, the assembly does not require readthrough of a translational reading frame shift or stop codon, common mechanisms used by retroelements to express Gag in molar excess of the polymerase proteins. This study was designed to determine if Tf1 particles contain equal amounts of Gag and polymerase proteins or whether they contain the typical molar excess of Gag. After using two separate methods to calibrate the strength of our antibodies, we found that both S. pombe extracts and partially purified Tf1 particles contained a 26-fold molar excess of Gag relative to IN. Knowing that Gag and IN are derived from the same Tf1 primary translation product, we concluded that the excess Gag most likely resulted from specific degradation of IN. We obtained evidence of regulated IN degradation in comparisons of Tf1 protein extracted from log-phase cells and that extracted from stationary-phase cells. The log-phase cells contained equal molar amounts of Gag and IN, whereas cells approaching stationary phase rapidly degraded IN, leaving an excess of Gag. Analysis of the reverse transcripts indicated that the bulk of reverse transcription occurred within the particles that possess a molar excess of Gag.

Base Sequence↗

PubMed2002

Neuronal overexpression of "readthrough" acetylcholinesterase is associated with antisense-suppressible behavioral impairments.

Molecular origin(s) of the diverse behavioral responses to anticholinesterases were explored in behaviorally impaired transgenic (Tg) FVB/N mice expressing synaptic human acetylcholinesterase (hAChE-S). Untreated hAChE-S Tg, unlike naïve FVB/N mice, presented variably intense neuronal overexpression of the alternatively spliced, stress-induced mouse "readthrough" mAChE-R mRNA. Both strains displayed similar diurnal patterns of locomotor activity that were impaired 3 days after a day-to-night switch. However, hAChE-S Tg, but not FVB/N mice responded to the circadian switch with irregular, diverse bursts of increased locomotor activity. In social recognition tests, controls displayed short-term recognition, reflected by decreased exploration of a familiar, compared to a novel juvenile conspecific as well as inverse correlation between social recognition and cortical and hippocampal AChE specific activities. In contrast, transgenics presented poor recognition, retrievable by tetrahydroaminoacridine (tacrine, 1.5 mg kg(-1)). Tacrine's effect was short-lived (24 h) suppression of the abnormal social recognition pattern in transgenics. Efficacy of antisense treatment was directly correlated with AChE-R levels and the severity of the impaired phenotype, being most apparent in transgenics presenting highly abnormal pre-treatment behavior. These findings demonstrate that neuronal AChE-R overproduction is involved in various behavioral impairments and anticholinesterase responses, and point to the antisense strategy as a potential approach for re-establishing cholinergic balance.

Acetylcholinesterase↗

PubMed1994

Synthesis and processing in vivo of the novel mouse thyrotropin beta-presubunit that contains an NH2-terminal extension sequence.

Expression of the single mouse TSH beta gene gives rise to multiple mRNAs, and we have previously shown that in vitro, one of these mRNAs gives rise to a novel TSH beta-presubunit due to initiation of translation at an in-frame start site unique to this mRNA which is up-stream of the normal start site. The novel presubunit contains a 17-amino acid NH2-terminal extension sequence compared to the normal presubunit. Although this extension sequence does not have the characteristics of a normal signal sequence, the novel TSH beta-presubunit was processed in vitro by microsomal membranes. In this study we have examined the translation product of this mRNA in intact cells and whether in vivo it gives rise to a processed secreted TSH beta-subunit that has an NH2-terminal sequence different from that of the established TSH beta-subunit. Firstly, mRNAs encoding alpha-presubunit and either the normal or novel TSH beta-presubunit were microinjected into Xenopus oocytes, and it was found that immunoprecipitable TSH dimer was secreted into the medium regardless of the mRNA used for TSH beta-subunit synthesis. However, less TSH was obtained when the TSH beta-subunit was derived from the extended TSH beta-presubunit. Secondly, when COS cells were transiently transfected with plasmids expressing alpha-presubunit and either the normal or novel TSH beta-presubunit, secreted TSH was obtained when the TSH beta-subunit was derived from either presubunit. TSH dimer was also obtained when the TSH beta-presubunit was derived from a mRNA encoding the extended presubunit in which the down-stream AUG had been eliminated by site-specific mutagenesis. This demonstrated that the up-stream translation start site was used in the intact cell and that secreted TSH beta-subunit was derived from the extended presubunit and not from normal presubunit resulting from translational readthrough to the down-stream AUG. When secreted TSH beta-subunits derived from the normal and extended TSH beta-presubunits were digested with endoproteinase LysC, the NH2-terminal fragments were similar in size, suggesting that the NH2-terminal extension had little if any effect on the site of cleavage by signal peptidase. Our data, therefore, demonstrate that the longer TSH beta-presubunit is synthesized in vivo and strongly suggest that it is processed in the intact cell to give a mature secreted TSH beta-subunit indistinguishable from that derived from the normal TSH beta-presubunit.

Amino Acid Sequence↗

PubMed1998

Uncoupled expression of p33 and p92 permits amplification of tomato bushy stunt virus RNAs.

Tomato bushy stunt virus (TBSV) is a plus-sense RNA virus which encodes a 33-kDa protein in its 5'-most open reading frame (ORF). Readthrough of the amber stop codon of the p33 ORF results in the production of a 92-kDa fusion protein. Both of these products are expressed directly from the viral genome and are suspected to be involved in viral RNA replication. We have investigated further the roles of these proteins in the amplification of viral RNAs by using a complementation system in which p33 and p92 are expressed from different viral RNAs. Our results indicate that (i) both of these proteins are necessary for viral RNA amplification; (ii) translation of these proteins can be uncoupled while maintaining amplification of viral RNAs; (iii) if compatibility requirements exist between p33 and p92, they are not exceptionally strict; and (iv) the C-terminal approximately 6% of p33 is necessary for its functional activity. Interestingly, no complementation was observed when a p33-encoding replicon containing a deletion of a 3'-located segment, region 3.5, was tested. However, when 5'-capped transcripts of the same replicon were analyzed, complementation allowing for RNA amplification was observed. This ability to compensate functionally for the absence of region 3.5 by the addition of a 5' cap suggests that this RNA segment may act as a translational enhancer for the expression of virally encoded products.

Gene Amplification↗

PubMed1989

Mutagenesis of the in-frame opal termination codon preceding nsP4 of Sindbis virus: studies of translational readthrough and its effect on virus replication.

Sindbis virus (SIN) contains an in-frame opal termination codon in the nonstructural protein-coding region separating nsP3 and nsP4 and provides a useful tool to study the readthrough phenomenon of the termination codon in host cells and its role in viral replication. We have changed the opal codon by site-directed mutagenesis of a full-length SIN cDNA clone to either sense amino acids (serine, tryptophan, or arginine) or the other two translation termination codons (amber or ochre). Transcripts from all of the mutant cDNA clones were infectious when used to transfect chicken embryo fibroblasts. The resulting progeny virus stocks were then used to study the effects of these mutations on viral protein and RNA synthesis, growth properties, host range, and fitness compared with the parental strain. None of the mutants showed temperature sensitivity in plaquing efficiency or plaque morphology on chicken embryo fibroblast monolayers. Relative to the wild-type parent, the mutants containing sense replacements overproduced nsP34 but not nsP4 and made slightly decreased levels of nsP3, with a delay in its appearance. This indicates that the cleavage separating nsP3 and nsP4 occurs in these mutants and also that the level of nsP4 is not regulated solely by readthrough of the opal codon. The amber and ochre mutants produced decreased levels of nsP34, and the ochre mutant grew significantly more slowly than the other mutants or wild-type virus. For all five mutants, RNA synthesis early in infection was inhibited compared with that of the parental virus. This effect was apparent at multiplicities of infection of 20 PFU per cell but not at 100 PFU per cell. Using in situ hybridization to distinguish between mutant and wild-type plaques, we have studied the behavior of the serine mutant in a high-multiplicity growth competition experiment with wild-type virus. The wild-type virus eventually outcompeted the mutant after several passages, and these results indicate that this mutation has resulted in effects that are at least partially cis acting. Furthermore, by studying the growth, plaque formation, and protein synthesis of the mutants in various cell types, we have observed host range effects of the mutations, especially in mosquito and human cells. In addition, we have demonstrated, at least indirectly, that opal, amber, and ochre termination codons in the SIN nucleotide context can be suppressed in cultured cells of chicken, human, hamster, and mosquito origin.

Animals↗

PubMed1998

Threshold number of provirus copies required per cell for efficient virus production and interference in moloney murine leukemia virus-infected NIH 3T3 cells.

The gag-pol readthrough mutant of Moloney murine leukemia virus, MLV-B(CAG) (T. Odawara, H. Yoshikura, M. Oshima, T. Tanaka, D. S. Jones, F. Nemoto, Y. Kuchino, and A. Iwamoto, J. Virol. 65:6376-6379, 1991), was poorly complemented by a mutant encoding only Gag. This is because with all the genetic elements necessary for env expression present in MLV-B(CAG), insufficient Env protein was produced by the cells expressing MLV-B(CAG) for efficient virus production. Since the env mRNA expression per provirus in the MLV-B(CAG)- and wild-type-MLV-producing cells were the same and since the cells expressing the former contained eightfold fewer proviral copies, the insufficient Env expression by the former was found to be due to insufficient proviral copies in the cells. Examination of the cell clones having various proviral copies of Deltawt MLV (M. Oshima, T. Odawara, T. Matano, H. Sakahira, Y. Kuchino, A. Iwamoto, and H. Yoshikura, J. Virol. 70:2286-2295, 1996) showed that mRNA level was proportional to the number of proviral copies while interference and virus production followed a sigmoid curve with a sharp rise at the threshold number of proviral copies of around four per cell. Multicycle infection probably continues until the threshold level of proviral copies is attained in natural infection too.

3T3 Cells↗

PubMed1989

Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription.

Human rRNA precursor from normal or stressed HeLa cells were studied by S1 nuclease mapping of unlabeled RNA and by antisense RNase mapping of RNA from cells that had been labeled in vivo with [32P]PO4. Heating cells to 43 degrees C decreased the amount of newly synthesized rRNA to less than 5% of the control level and led to greater than 95% inhibition of transcription termination at a region 355 to 362 nucleotides downstream of the 3' end of 28S rRNA, with readthrough continuing into the next transcription unit. Heating of cells to 42 degrees C led to 60% inhibition of termination at this site; 50% of transcripts that extended into the nontranscribed spacer ended in a region 200 to 210 nucleotides upstream of the polymerase I (Pol I) initiation site. This is presumed to be the human upstream transcription termination site because of the absence of RNAs with a 5' end corresponding to this region, the location relative to the Pol I initiation site (which is similar to the location of upstream terminators in other species), and the fact that it is 15 to 25 nucleotides upstream of the sequence GGGTTGACC, which has an 8-of-9 base identity with the sequence 3' of the downstream termination site. Surprisingly, treatment of cells with sodium arsenite, which also leads to the induction of a stress response, did not inhibit termination. Pol I initiation was decreased to the same extent as termination, which lends support to the hypothesis that termination and initiation are coupled. Although termination was almost completely inhibited at 43 degrees C, the majority of the recently synthesized rRNAs were processed to have the correct 3' end of 28S. This finding suggests that 3'-end formation can involve an endonucleolytic cut and is not solely dependent on exonucleolytic trimming of correctly terminated rRNAs.

Animals↗

PubMed2003

A Mg2+-dependent RNA tertiary structure forms in the Bacillus subtilis trp operon leader transcript and appears to interfere with trpE translation control by inhibiting TRAP binding.

Expression of the trpEDCFBA operon of Bacillus subtilis is regulated by transcription attenuation and translation control mechanisms. In each case, binding of the trp RNA-binding attenuation protein (TRAP) to the untranslated trp leader transcript mediates conformational changes in the RNA secondary structure. We examined the structure of the trp leader readthrough RNA in the absence of TRAP. Using chemical and enzymatic probes, the secondary structure of the trp leader RNA was found to be similar to predicted models. In addition, this RNA was found to adopt a Mg(2+)-dependent, long-range tertiary interaction under physiological monovalent salt conditions. Formation of this tertiary structure does not require significant changes in the preformed secondary structure. Enzymatic probing of the RNA in the presence of competitor DNA oligonucleotides that were designed to disrupt the predicted tertiary structure allowed identification of the interacting partners as the single-stranded portion of the purine-rich TRAP binding target and a large downstream pyrimidine-rich internal loop. UV cross-linking experiments utilizing 5'-p-azidophenacyl-containing transcripts revealed a Mg(2+)-dependent cross-link. Mapping of this cross-link provided evidence that the single-stranded segment of the TRAP binding site is in close proximity to the internal loop. Results from UV melting experiments with wild-type and mutant trp leader transcripts suggested a likely base-pairing register for the tertiary structure. Filter-binding studies demonstrated that the addition of Mg(2+) inhibits TRAP binding, which may be partially due to the effect of Mg(2+) on RNA tertiary structure formation. Results from expression studies using trpE'-'lacZ translational fusions and RNA-directed cell-free translation experiments suggest that the Mg(2+)-dependent tertiary structure inhibits TRAP's ability to regulate translation of trpE.

Bacillus subtilis↗

PubMed1989

Sequences linked to prokaryotic promoters can affect the efficiency of downstream termination sites.

The efficiency of transcription termination at certain well-defined prokaryotic rho-independent terminators depends on the promoter unit from which transcription is initiated. Some promoter units allow substantial readthrough of strong termination signals, a phenomenon we term "factor-independent antitermination". This observation is not easily explained by current models for prokaryotic terminator function that consider the terminator to be a "cassette" involving only sequences and RNA transcript structures in the immediate region of the terminator or directly upstream. When transcription is carried out in vitro employing only purified Escherichia coli RNA polymerase, up to 20 times as many RNA polymerase molecules pass through a particular terminator when transcription is initiated from the E. coli tac promoter unit, as compared to transcription initiated from the T7A1 or rrnB P2 promoters. This effect cannot be attributed to antitermination factors separate from the core RNA polymerase. Similar differences in termination efficiency are found for the same promoters in vivo. These termination differences are affected by sequences just downstream from the start site for transcription, including those in the +25 region of the nascent transcript. These early transcribed sequences can confer factor-independent antitermination onto a heterologous promoter, but only when the sequences are precisely positioned relative to the start site for transcription. We have considered several possible models to explain how early transcribed sequences might affect termination, including those in which the 5' end of the transcript interacts with either the terminator RNA or the polymerase. We favor an alternative model in which these sequences interact with the core RNA polymerase to convert the enzyme from a termination-proficient state (T-state) to a conformation resistant to termination (R-state). Such enzyme conformations may be an important component of factor-dependent antitermination systems.

Base Sequence↗