Single Stop Research Articles

Unusual tubulins with deviant genetic code in the hypotrich ciliate Euplotidium itoi

We characterized nine macronuclear tubulin gene minichromosomes in the hypotrich ciliate Euplotidium itoi: two α‐tubulin genes, five β‐tubulin genes, and two γ‐tubulin genes. In particular, one peculiar β‐tubulin gene was identified. It shows only 81.5% amino acid similarity to the other characterized β‐tubulin types and an UAG stop codon in frame. Based on this result we started to analyse stop codon usage in Euplotidium itoi macronuclear tubulin genes and some unrelated macronuclear minichromosomes. Most of the ciliates independently evolved diverse non‐canonical genetic codes. Differences to the standard code consist in the reassignment of one or two of the three standard stop codons to encode a particular amino acid. Class Spirotrichea represents a particular case comprising two groups of organisms that do not share any stop codon. In oxytrichids, UAR is translated as glutamine and UGA is the single stop codon. This is in contrast to the genus Euplotes, close relatives of Euplotidium, in which UAR encodes a stop signal and UGA is translated as cysteine. Fourteen complete genes were characterized in Euplotidium itoi; in all cases UAA revealed as the single triplet used to code a stop, UGA was never and UAG rarely observed in frame. The UAG stop codon was observed in the peculiar type 3 β‐tubulin gene, in two dynein partial sequences and in some other putative genes. We could confirm the active expression of three of these genes, the unusual β‐tubulin, a putative lipase and a possible steroid‐binding protein, by RT‐PCR. Finally, a detailed analysis on the structure of the macronuclear tubulin genes, including nucleotide and amino acid differences among tubulin gene paralogs, polyadenylation sites, putative chromosome fragmentation sites, ATGC‐content in coding and non‐coding regions, telomere sequence and length will be presented.

Termination and read-through proteins encoded by genome segment 9 of Colorado tick fever virus

Genome segment 9 (Seg-9) of Colorado tick fever virus (CTFV) is 1884 bp long and contains a large open reading frame (ORF; 1845 nt in length overall), although a single in-frame stop codon (at nt 1052-1054) reduces the ORF coding capacity by approximately 40 %. However, analyses of highly conserved RNA sequences in the vicinity of the stop codon indicate that it belongs to a class of 'leaky terminators'. The third nucleotide positions in codons situated both before and after the stop codon, shows the highest variability, suggesting that both regions are translated during virus replication. This also suggests that the stop signal is functionally leaky, allowing read-through translation to occur. Indeed, both the truncated 'termination' protein and the full-length 'read-through' protein (VP9 and VP9', respectively) were detected in CTFV-infected cells, in cells transfected with a plasmid expressing only Seg-9 protein products, and in the in vitro translation products from undenatured Seg-9 ssRNA. The ratios of full-length and truncated proteins generated suggest that read-through may be down-regulated by other viral proteins. Western blot analysis of infected cells and purified CTFV showed that VP9 is a structural component of the virion, while VP9' is a non-structural protein.

A thermal analysis of a new railway brake concept using liquid cooling

The aim of this study is to evaluate the performances of the energy dissipation of a new railway disc brake concept, intended for an urban exploitation. Because of the increase in the rates of trains and subways as well as the number of passengers, brakes have become increasingly a major safety component. In urban exploitation, these systems are subjected to multiple braking actions and have operational limits. A new concept based on a liquid-cooled system is suggested to decrease the energy storage in the disc. A numerical model of this new concept was realized and its performances were estimated by comparing temperature evolution at the friction interface with that obtained with the classical brake. The temperatures reached on a single stop with the cooled concept are higher than those of a classical brake. However, an important temperature decrease is observed at the end of braking so that, for successive brakings, the liquid-cooling principle causes an overall decrease in temperature. Moreover, the use of a material with a high thermal effusivity increases the part of the flow generated at the interface transmitted to the pad and thus reduces the surface temperature.

Period-doubling bifurcations and routes to chaos of the vibratory systems contacting stops

A two-degree-of-freedom vibratory system contacting a single rigid stop is considered. The stop leads motions with repeat impacts, i.e., vibro-impacts. Such models play an important role in the studies of mechanical systems with amplitude constraining stops. Some non-typical routes to chaos, via period-doubling bifurcations of periodic motions with one impact, are studied. The period-doubling cascades of periodic orbits with one impact are usually discontinuous in the transition of periodic impact to chaos, in which there exist grazing bifurcation, torus bifurcation or Hopf bifurcation associated with multi-impact motions with higher period. A two-degree-of-freedom system having symmetrically placed rigid stops and subjected to periodic excitation is considered also. The system may exhibit more complicated dynamical behavior in the route to chaos via pitchfork bifurcation of period-one double-impact symmetrical motion. The routes to chaos, appearing in the vibro-impact systems, are qualitatively different form the typical period-doubling route to chaos in the usual consecutive maps.

The soybean retroelement SIRE1 uses stop codon suppression to express its envelope-like protein.

The soybean SIRE1 family of Ty1/copia retrotransposons encodes an envelope-like gene (env-like). We analysed the DNA sequences of nine SIRE1 insertions and observed that the gag/pol and env-like genes are in the same reading frame and separated by a single UAG stop codon. The six nucleotides immediately downstream of the stop codon conform to a degenerate nucleotide motif, CARYYA, which is sufficient to facilitate stop codon suppression in tobacco mosaic virus. In vivo stop codon suppression assays indicate that SIRE1 sequences confer leakiness to the UAG stop codon at an efficiency of 5%. These data suggest that SIRE1 retro-elements use translational suppression to express their envelope-like protein; this is in contrast with all characterized retroviruses, which express the envelope protein from a spliced genomic messenger RNA.

A small reservoir of disabled ORFs in the yeast genome and its implications for the dynamics of proteome evolution

We surveyed the sequenced Saccharomyces cerevisiae genome (strain S288C) comprehensively for open reading frames (ORFs) that could encode full-length proteins but contain obvious mid-sequence disablements (frameshifts or premature stop codons). These pseudogenic features are termed disabled ORFs (dORFs). Using homology to annotated yeast ORFs and non-yeast proteins plus a simple region extension procedure, we have found 183 dORFs. Combined with the 38 existing annotations for potential dORFs, we have a total pool of up to 221 dORFs, corresponding to less than ∼3 % of the proteome. Additionally, we found 20 pairs of annotated ORFs for yeast that could be merged into a single ORF (termed a mORF) by read-through of the intervening stop codon, and may comprise a complete ORF in other yeast strains. Focussing on a core pool of 98 dORFs with a verifying protein homology, we find that most dORFs are substantially decayed, with ∼90 % having two or more disablements, and ∼60 % having four or more. dORFs are much more yeast-proteome specific than live yeast genes (having about half the chance that they are related to a non-yeast protein). They show a dramatically increased density at the telomeres of chromosomes, relative to genes. A microarray study shows that some dORFs are expressed even though they carry multiple disablements, and thus may be more resistant to nonsense-mediated decay. Many of the dORFs may be involved in responding to environmental stresses, as the largest functional groups include growth inhibition, flocculation, and the SRP/TIP1 family. Our results have important implications for proteome evolution. The characteristics of the dORF population suggest the sorts of genes that are likely to fall in and out of usage (and vary in copy number) in a strain-specific way and highlight the role of subtelomeric regions in engendering this diversity. Our results also have important implications for the effects of the [PSI+] prion. The dORFs disabled by only a single stop and the mORFs (together totalling 35) provide an estimate for the extent of the sequence population that can be resurrected readily through the demonstrated ability of the [PSI+] prion to cause nonsense-codon read-through. Also, the dORFs and mORFs that we find have properties (e.g. growth inhibition, flocculation, vanadate resistance, stress response) that are potentially related to the ability of [PSI+] to engender substantial phenotypic variation in yeast strains under different environmental conditions. (See genecensus.org/pseudogene for further information.)

PERIODIC MOTIONS AND GLOBAL BIFURCATIONS OF A TWO-DEGREE-OF-FREEDOM SYSTEM WITH PLASTIC VIBRO-IMPACT

A two-degree-of-freedom (d.o.f.) impact system with proportional damping is considered. The maximum displacement of one of the masses is limited to a threshold value by a rigid wall, which gives rise to a non-linearity in the system. A limiting case of a dynamical problem arising in the mechanical systems with amplitude constraints is investigated. For the perfectly plastic vibro-impact case, dynamics of a two-degree-of-freedom vibratory system contacting a single stop is represented by a three-dimensional map. Existence and stability of period n single-impact motions are analyzed by analytical and numerical methods. It is shown that the vibro-impact system may exhibit two different types of periodic motions due to the piecewise property of the map. Transitions of two types of period n single-impact motions are demonstrated. The singularities of the Poincaré map, caused by grazing boundary motion of the impact oscillator, are considered. Due to the piecewise discontinuities and singularities of the map, the vibro-impact system is shown to undergo period-doubling bifurcations followed by complex sequence of transitions, in which the period-doubling cascades do not occur and extremely long-periodic and chaotic motions are generated directly with the motions with grazing boundary occurring. Finally, the influence of system parameters on periodic impacts and global bifurcations is discussed.

Redundancy gain in the stop-signal paradigm: Implications for the locus of coactivation in simple reaction time.

The authors carried out 2 experiments designed to cast light on the locus of redundancy gain in simple visual reaction time by using a stop-signal paradigm. In Experiment 1, the authors found that single visual stimuli were more easily inhibited than double visual stimuli by an acoustic stop signal. This result is in keeping with the idea that redundancy gain occurs prior to the ballistic stage of the stop-signal task. In Experiment 2, the authors found that the response to an acoustic go signal was more easily inhibited by a double than by a single visual stop signal. This result provides conclusive evidence for a redundancy gain in the stop process--in a process that does not involve a motor response but rather its inhibition.

Results of a Clinical Comprehensive Stop Smoking Clinic Using Nicotine Patch or Bupropion SR Among Veteran Smokers

PURPOSE: Cigarette smoking is the leading preventable cause of death in the United States. Several medications are efficacious for smoking cessation in clinical trials involving healthy volunteers, but little information is available regarding comparative effectiveness in clinical programs. The purpose of this evaluation was to compare the effectiveness of two smoking cessation medications when combined with behavioral counseling. METHODS: Over a one-year period outpatient veterans were treated with either the nicotine patch (14–35 mg once daily) or bupropion SR (150 mg twice daily) for 8 weeks at a single outpatient stop smoking clinic. The behavioral program included 4 1/2 hours of cognitive-behavioral counseling, brand-switching, self-help manual, relaxation tape, and a cigarette substitute. Patients attended the clinic every other week for 3 visits, and were contacted by telephone 3 months after the quit date. Patients who were unavailable for follow-up were assumed to be smoking. RESULTS: Two-hundred seventy-two patients received treatment with either nicotine patch (n = 168) or bupropion SR (n = 104) by clinic assignment. The mean age was 53.0 years (SD = 11.6), 55.9% were Caucasian; 42.3% were African-American, 99% were male. The baseline mean cigarettes smoked per day was 27.8 cigarettes (SD = 15.7), the mean Fagerstrom Test for Nicotine Dependence (FTND) was 6.3 (SD = 2.2), the mean exhaled carbon monoxide was 22.3 (SD = 10.5), the mean Beck Depression scale was 12.5 (SD = 10.9), the mean Beck Anxiety scale was 12.6 (12.0). Thirty-six percent of patients had previously used the nicotine patch, 30% had previously used the nicotine gum. Fifty-three percent had hypertension, 22.5% reported a history of heart disease. The treatment groups were similar except that patients in the bupropion SR group had significantly higher previous nicotine patch use, and significantly lower FTND scores. Telephone follow-up at 3 months was completed in 59.8% of patients, and one-week point prevalence abstinence rates were 25.5% for the bupropion SR group, and 24.4% for the nicotine patch group (between group difference = 1.1%, p = NS; 95% CI for the difference: -9.4% to 11.6%). Abstinence was not related to baseline anxiety or depression scores. On a 0–5 ordinal scale, patients rated the helpfulness of classes a mean = 4.1 (SD = 0.98), self-help book = 3.3 (SD = 1.3), the cigarette substitute = 2.4 (SD = 1.5), and the relaxation tape = 2.3 (SD = 1.7). CONCLUSION: We conclude that buproprion SR and nicotine patch have similar effectiveness when used for 8 weeks in combination with a comprehensive behavioral program. Given the relatively low abstinence rates, more intensive treatment appears in order to enhance abstinence rates in this clinical population.

Single stop production at hadron colliders

We analyze the production of a single top squark at hadron colliders: p p ̄ /pp→ t ̃ 1+X . The total cross sections and the transverse momentum distributions are presented in next-to-leading order QCD. The higher-order corrections render the predictions theoretically stable with respect to variations of the factorization and renormalization scales. The transverse momentum distribution is resummed to estimate effects of the small transverse momentum regime on possible analyses. Since the corrections increase the cross sections and reduce the theoretical uncertainty, the discovery range for these particles is extended in the refined analysis.

Interactions of ribosome nascent chain complexes of the chloroplast-encoded D1 thylakoid membrane protein with cpSRP54.

The mechanisms of targeting, insertion and assembly of the chloroplast-encoded thylakoid membrane proteins are unknown. In this study, we investigated these mechanisms for the chloroplast-encoded polytopic D1 thylakoid membrane protein, using a homologous translation system isolated from tobacco chloroplasts. Truncated forms of the psbA gene were translated and stable ribosome nascent chain complexes were purified. To probe the interactions with the soluble components of the targeting machinery, we used UV-activatable cross-linkers incorporated at specific positions in the nascent chains, as well as conventional sulfhydryl cross-linkers. With both cross-linking approaches, the D1 ribosome nascent chain was photocross-linked to cpSRP54. cpSRP54 was shown to interact only when the D1 nascent chain was still attached to the ribosome. The interaction was strongly dependent on the length of the nascent chain that emerged from the ribosome, as well as the cross-link position. No interactions with soluble SecA or cpSRP43 were found. These results imply a role for cpSRP54 in D1 biogenesis.

Skipper, an LTR retrotransposon of Dictyostelium.

The complete sequence of a retrotransposon from Dictyostelium discoideum , named skipper , was obtained from cDNA and genomic clones. The sequence of a nearly full-length skipper cDNA was similar to that of three other partially sequenced cDNAs. The corresponding retrotransposon is represented in approximately 15-20 copies and is abundantly transcribed. Skipper contains three open reading frames (ORFs) with an unusual sequence organization, aspects of which resemble certain mammalian retroviruses. ORFs 1 and 3 correspond to gag and pol genes; the second ORF, pro, corresponding to protease, was separated from gag by a single stop codon followed shortly thereafter by a potential pseudoknot. ORF3 (pol) was separated from pro by a +1 frameshift. ORFs 2 and 3 overlapped by 32 bp. The computed amino acid sequences of the skipper ORFs contain regions resembling retrotransposon polyprotein domains, including a nucleic acid binding protein, aspartyl protease, reverse transcriptase and integrase. Skipper is the first example of a retrotransposon with a separate pro gene. Skipper is also novel in that it appears to use stop codon suppression rather than frameshifting to modulate pro expression. Finally, skipper and its components may provide useful tools for the genetic characterization of Dictyostelium.

Analysis of a 32.8 kb segment of yeast chromosome IV reveals 21 open reading frames, including TPS2, PPH3, RAD55, SED1, PDC2, AFR1, SSS1, SLU7 and a tRNA for arginine.

We report the nucleotide sequence of a 32.8 kb DNA segment from the right arm of Saccharomyces cerevisiae chromosome IV. The sequence contains 20 open reading frames (ORFs) longer than 300 bp as well as the 240 bp gene coding for the essential SSS1 secretory protein. Nine ORFs previously totally or partially sequenced (TPS2, PPH3, RAD55, SED1, PDC2, AFR1, SSS1, SLU7 and D4478) are presented, as well as the transmembrane protein D4405, the leucine zipper containing D4495 and a new tRNA for arginine. D4456 and D4461 are separated by a single in-frame stop codon only. The other five ORFs show no particular features or significant homology.

Regulatory autonomy and molecular characterization of the Drosophila out at first gene.

Our previous work has shown that the expression of the Drosophila decapentaplegic (dpp) gene in imaginal disks is controlled by a 30 kb array of enhancers located 3' of the dpp coding region. Here, we describe the cloning and characterization of out at first (oaf), a gene located near this enhancer region. Transcription of oaf results in three classes of alternatively polyadenylated RNAs whose expression is developmentally regulated. All oaf transcripts contain two adjacent open reading frames separated by a single UGA stop codon. Suppression of the UGA codon during translation, as seen previously in Drosophila, could lead to the production of different proteins from the same RNA. During oogenesis, oaf RNA is expressed in nurse cells of all ages and maternally contributed to the egg. During embryonic development, zygotic transcription of the gene occurs in small clusters of cells in most or all segments at the time of germband extension and subsequently in a segmentally repeated pattern in the developing central nervous system. The gene is also expressed in the embryonic, larval and adult gonads of both sexes. We also characterize an enhancer trap line with its transposon inserted within the oaf gene and use it to generate six recessive oaf mutations. All six cause death near the beginning of the first larval instar, with two characterized lines showing nervous system defects. Last, we discuss our data in light of the observation that the enhancers controlling dpp expression in the imaginal disks have no effect on the relatively nearby oaf gene.

A novel family of cathepsin L-like (CTSLL) sequences on human chromosome 10q and related transcripts.

We have isolated a human genomic DNA cosmid clone while screening for the cathepsin L gene that, when sequenced, revealed close similarity with but significant differences from cDNA sequences that have been reported for cathepsin L (CTSL). The clone bears a novel sequence that shows 88% identity to the coding regions of the cathepsin L gene and a similar exon arrangement. We have called this sequence the "human cathepsin L-like gene 1" (CTSLL1). Translating putative exon sequences reveals a single premature stop codon; therefore no functional products are likely to arise from this gene. Fluorescence in situ hybridization (FISH) studies mapped the clone to chromosome 10q. Somatic cell hybrid mapping confirmed the location of CTSLL1 to human chromosome 10 distinct from the cathepsin L locus (CTSL) on chromosome 9. Furthermore, the FISH mapping studies show that a family of at least three related sequences exists on chromosome 10q, similar to the pattern of duplicated glutamate dehydrogenase (GLUD) gene loci reported on 10q. Using PCR and sequencing with genomic DNA samples, we have identified two additional novel related sequences (CTSLL2 and CTSLL3), and by PCR analysis of cDNA samples we have identified corresponding transcripts. Comparison of changes between our CTSLL1 sequence and the cathepsin L gene at mutation insensitive sites suggests that the two sequences arose from a duplication event 40-50 million years ago, and therefore at the time of divergence of early primates.

Acoustic manifestations of mora timing in Japanese

Segment durations in 20 minimal-paired words in Japanese were examined to identify regular patterns of acoustic manifestations of mora timing in Japanese. The results indicate that segments consistently stretch or compress, providing temporal compensation within a mora and between moras, to attain targeted length at the word level. A segment is 11% longer in the pre-geminate-stop position than in the pre-single-stop position; in the word final position, a segment is 9% shorter following a geminate stop than a segment following a single stop; and [t] in the same phonotactic environment shows varied durations according to different moraic conditions. Fricatives followed by a devoiced vowel are considerably longer than any other inherently long, nonmoraic segments, and are comparable to moraic segments and the average mora duration. Syllable durations are not isochronous, varying by as much as 50%. The word duration is affected more by the number of component sounds and their inherent durations in two-mora than in three-or four-mora words. When the word durations are converted into ratios, they are markedly similar to the mora values. The results suggest a timing control mechanism adjusting segments to obtain targeted word durations. These phenomena describe mora timing in Japanese.

Changes in Spinal Reflex Excitability in a Countermanded Timed Response Task

Subjects (N = 8) performed a timed response task in which they attempted to synchronize an impulsive foot-press response with the last in a series of four regularly spaced tones. In Experiment 1, the response was countermanded on one third of the trials (stop trials) by a stop signal that appeared at a predetermined delay after the third tone. No stop signal appeared on the remaining trials (go trials). All subjects showed a systematic transition from withholding the response on stop trials in which the stop signal appeared shortly after the third tone to executing the response on trials in which a single stop signal delay had been chosen so that a response would be made on about 50% of the stop trials. We elicited Hoffmann (H) reflexes from the soleus muscle on all trials to determine whether the reflexes were augmented on occasions on which a response was prepared but withheld. Mean H-reflex amplitudes on go trials and on stop trials on which the response was executed were similar and showed a marked augmentation beginning about 250 ms before response onset; mean H-reflex amplitudes on stop trials on which the response was withheld showed less pronounced augmentation. Inspection of individual H-reflex amplitudes revealed that on stop trials on which the response was withheld the reflexes could be augmented to the same extent as on trials on which the response was executed. This dissociation of H-reflex augmentation and response execution shows that H-reflex augmentation reflects a controlled response process. Ballistic response processes therefore must be limited to a brief duration.

Three Tetrahymena tRNA(Gln) isoacceptors as tools for studying unorthodox codon recognition and codon context effects during protein synthesis in vitro.

Three glutamine tRNA isoacceptors are known in Tetrahymena thermophila. One of these has the anticodon UmUG which reads the two normal glutamine codons CAA and CAG, whereas the two others with CUA and UmUA anticodons recognize UAG and UAA, respectively, which serve as termination codons in other organisms. We have employed these tRNA(Gln)-isoacceptors as tools for studying unconventional base interactions in a mRNA- and tRNA-dependent wheat germ extract. We demonstrate here (i) that tRNA(Gln)UmUG suppresses the UAA as well as the UAG stop codon, involving a single G:U wobble pair at the third anticodon position and two simultaneous wobble base pairings at the first and third position, respectively, and (ii) that tRNA(Gln)CUA, in addition to its cognate codon UAG, reads the UAA stop codon which necessitates a C:A mispairing in the first anticodon position. These unorthodox base interactions take place in a codon context which favours readthrough in tobacco mosaic virus (TMV) or tobacco rattle virus (TRV) RNA, but are not observed in a context that terminates zein and globin protein synthesis. Furthermore, our data reveal that wobble or mispairing in the middle position of anticodon-codon interactions is precluded in either context. The suppressor activities of tRNAs(Gln) are compared with those of other known naturally occurring suppressor tRNAs, i.e., tRNA(Tyr)G psi A and tRNA(Trp)CmCA. Our results indicate that a 'leaky' context is neither restricted to a single stop codon nor to a distinct tRNA species.

Cloning and sequence analysis of a hemolysin-encoding gene from Pseudomonas paucimobilis.

We report the cloning, expression and nucleotide (nt) sequence of a beta-hemolysin-encoding gene, termed hlyA, from Pseudomonas paucimobilis. A genomic DNA library of the pseudomonad was constructed in Escherichia coli using the plasmid vector, pUC19. The hlyA gene was cloned by screening for a beta-hemolytic phenotype in E. coli transformants and was mapped to a 1100-bp PstI-SmaI fragment. The nt sequence analysis of the 1100-bp insert revealed a 789-bp open reading frame which is preceded by a 10-nt purine-rich sequence with a possible ribosome-binding site of GGA. The ORF terminates with a single UGA stop codon and is immediately followed by a large inverted repeat with 27-bp arms which may serve as a Rho-factor-independent transcriptional terminator. The hlyA gene codes for a protein of 263 amino acids (aa) residues with a deduced relative molecular mass (M(r)) of 29,695 and a predicted pI value of 11.5. Expression of hlyA from recombinant DNA in E. coli occurred regardless of insert orientation in the vector and produced a 29-kDa protein. Confirmation of P. paucimobilis as the source of the cloned hlyA gene was determined by DNA hybridization. A search of various nt and aa sequence databases revealed no homologues to hlyA or its encoded protein.

Kelch encodes a component of intercellular bridges in Drosophila egg chambers

Oocyte maturation in Drosophila is supported by a cluster of 15 germline-derived nurse cells whose cytoplasm is transported into the oocyte through intercellular bridges called ring canals. Kelch was isolated as a female sterile mutation affecting cytoplasm transport. We have cloned the kelch gene and found that it encodes an unusual transcript containing two open reading frames (ORF1 and ORF2) separated by a single UGA stop codon. At least two protein products are made from the kelch mRNA: a short protein from ORF1 and a longer protein from both ORF1 and ORF2 as a result of partial suppression of the UGA codon. The kelch ORF1 product is conserved, and antibodies directed against it are localized specifically to ring canals. Our results suggest that kelch produces a component of ring canals that regulates the flow of cytoplasm between cells.