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J Gallant

Publications and source records attributed to J Gallant.

At least 37 records · Page 2Linked to original sources
PubMed1988

On the mechanism of ribosomal frameshifting at hungry codons.

In a few, rather rare cases, frameshift mutant alleles are phenotypically suppressed during limitation for particular aminoacyl-tRNA species. The simplest interpretation is compensatory ribosome frameshifting at a "hungry" codon in the vicinity of the suppressed frameshift mutation. We have now tested this interpretation directly by obtaining amino acid sequence data on such a phenotypically suppressed protein. We used a plasmid-borne lacZ gene, engineered to be in the (+) reading frame. Its background leakiness is increased by two orders of magnitude during lysyl-tRNA limitation. The enzyme made under this condition has the amino acid sequence expected from the DNA sequence up to the first lysine codon, then shifts in the (-) direction to recreate the correct lacZ reading frame. The lysine is replaced by serine, presumably due to cognate reading of an overlapping AGC codon displaced by one base to the 3' side of the AAG codon. When the 3' overlapping codon is AGA or AGG, there is no ribosome frameshifting; when it is AGU (read by the same serine tRNA) there is frameshifting, although less efficiently than in the case of AGC. The mechanism of cognate overlapping reading contradicts more elaborate models that two of the authors have suggested previously. However, the possibility remains that there is more than one mechanism of ribosome frameshifting at hungry codons.

Amino Acid Sequence↗
PubMed1988

Release factor competition is equivalent at strong and weakly suppressed nonsense codons.

We have compared the competition between strong or weak suppressor tRNAs and translational release factors (RF) at nonsense codons in the lacI gene of Escherichia coli. Using the F'lacIZ fusions developed by Miller and coworkers, UAG, UAA, and UGA codons at positions 189 and 220 were efficiently suppressed by plasmid-borne tRNA(trp) suppressors cognate to each nonsense triplet. Introduction of a compatible RF 1 plasmid competed at UAG and UAA but not UGA codons. An RF2 expressing plasmid competed at UAA and UGA but had little effect at UAG. Release factor competition against weak suppressors was measured using combinations of noncognate suppressors and nonsense codons. In each case, release factor plasmids behaved identically towards poorly suppressed codons as they did when the same codons were efficiently suppressed. The implications for these studies on the role of release factors in nonsense suppression context effects are discussed.

Codon↗
PubMed1988

Prototype volume-controlled neonatal/infant resuscitator.

Face mask or bag endotracheal tube manual ventilation of neonates and infants has relied primarily on the use of self-inflating resuscitation devices (SIRs). While SIRs have the advantage of being simple to use and, therefore, require minimal training, recent research has demonstrated significant drawbacks to these devices. These drawbacks have included their large physical size, the tendency of such devices to cause significant hyperventilation, and the extreme variability of pressures necessary to activate the pop-off valve safety feature. This latter problem is especially serious in the infant where large pressure variations may increase the potential for pulmonary barotrauma. In an attempt to minimize the disadvantages of SIRs, we developed a prototype volume-controlled resuscitation device (VCD). We then compared the VCD to the SIRs in a cat model using blinded volunteer health care personnel previously familiar with SIRs only. Ventilation of the cat with the VCD yielded arterial and end-tidal CO2 values more closely resembling the physiologic state at a lower mean airway pressure. The data suggest that a human trial of manual ventilation with the prototype VCD is warranted.

Animals↗
PubMed1988

Effects of release factor context at UAA codons in Escherichia coli.

In Escherichia coli, nonsense suppression at UAA codons is governed by the competition between a suppressor tRNA and the translational release factors RF1 and RF2. We have employed plasmids carrying the genes for RF1 and RF2 to measure release factor preference at UAA codons at 13 different sites in the lacI gene. We show here that the activity of RF1 and RF2 varies according to messenger context. RF1 is favored at UAA codons which are efficiently suppressed. RF2 is preferred at poorly suppressed sites.

Bacterial Proteins↗
PubMed1987

Nonsense suppression context effects in Escherichia coli bacteriophage T4.

Nonsense suppression by supE44 has been examined in a collection of 14 T4 gene 22 and gene 23 UAG mutants, for which the precise gene location is known. In concordance with previous studies, UAG followed by a pyrimidine was inefficiently suppressed. However, among positions with similar 3' nucleotides, there was considerable variation in suppression efficiency. The competition between supE44 and Release Factor 1 (RF 1) was also investigated following the introduction of a multicopy RF 1 plasmid. An inverse relationship between the efficiency of suppression and RF 1 competition was observed.

Codon↗
PubMed1987

Localization of small RNAs hybridizable to a B2 clone in the nuclear fraction of mouse cell lines.

An RNA polymerase III transcript, 7SK nuclear RNA, was found to bear sequence homology to the B2 class of highly repeated elements in the mouse genome. Northern blot hybridizations between the small RNAs and two B2 clones showed that only one of them (p49C8) hybridized to 7SK RNA. Both clones, however, hybridized to 4.5SI RNA as well as to a third class of nuclear RNA transcripts around 170 nucleotides long, whose levels were found to be greatly increased upon induction of transformation in a mouse 3T3 cell line transformed with a temperature-sensitive mutant of simian virus 40.

Animals↗
PubMed1986

Mapping and complementation studies of the gene for release factor 1.

In Escherichia coli the release factor 1 protein (RF1) recognizes and terminates translation at UAG and UAA codons. Using the technique of ColE1 plasmid integration in polA strains, we have mapped the cloned gene for RF1 to 27 min on the E. coli chromosome. This is the same location as that of the uar gene in which temperature-sensitive mutations increase the suppression of UAG and UAA alleles. In this study we proved that the uar mutation lies in the gene for RF1 by complementation of the uar phenotype with plasmids carrying the RF1 gene and by cloning the uar allele onto the RF1 plasmid by means of homologous recombination. In addition, complementation and P1 mapping data suggest that sueB is also a mutation in the same position as the RF1 gene. We propose that the gene for RF1 be named prfA after protein release factor.

Alleles↗
PubMed1983

Topology, organization, and function of the psi subunit in the F0 sector of the H+-ATPase of Escherichia coli.

The F1F0 H+-ATPase in membranes of Escherichia coli was amplified by heat induction of a lysogenic lambda-unc+ transducing phage. Inverted membrane vesicles were stripped of the F1 sector of the ATPase complex by washing with EDTA. The stripped membranes were treated with dithiobis(succimidylpropionate) to cross-link subunits of the F0 sector of the ATPase complex. After electrophoresis under nonreducing conditions in one dimension, cross-linked subunits were identified by off-diagonal electrophoresis in a second dimension following cleavage of the cross-linked products with beta-mercaptoethanol. A psi-psi dimer was the major cross-linked product identified. In addition, a chi-psi product and chi-psi2 product were identified. These results support the proposed chi-psi2 stoichiometry of subunits in F0. When the F1-stripped membranes were treated with trypsin, the psi subunit was rapidly degraded, whereas psi was protected from degradation when F1 was bound to the membrane. Trypsin-treated, stripped membranes, lacking an intact psi subunit, did not bind the F1 portion of the ATPase with high affinity. However, these trypsin-treated stripped membranes remained as permeable to protons as untreated stripped membranes, and the H+ conductivity was blocked by dicyclohexylcarbodiimide. These results indicate that the portion of the psi subunit exposed on the cytoplasmic face of the inner membrane is involved in the binding of the F1 portion of the ATPase, but is not necessary for H+ conduction mediated by the F0 sector of the complex.

Escherichia coli↗
PubMed1982

Nonsense suppression in aminoacyl-t-RNA limited cells.

A number of nonsense alleles of lacZ exhibit phenotypic suppression (as much as a sixteen-fold increase in leakiness) during partial limitation for certain aminoacyl-tRNA species in relA mutant cells. Each responsive allele has its individual pattern of response to limitation for one or more amino acids or aminoacyl-tRNA's. The phenotypic suppression occurs only during limitation, and ceases once limitation is reversed. Suppression is much reduced by the presence of the relA+ allele or an allele of rpsL which restricts ribosomal ambiguity. In one case, the suppressed product has been identified by radioimmune assay and gel electrophoresis, and is a full-length lacZ protomer. Mechanisms are discussed whereby aberrations of translation at codons calling for an aminoacyl-tRNA species in short supply might lead to readthrough of a nearby nonsense codon.

Escherichia coli↗
PubMed1982

An estimate of the global error frequency in translation.

Electrophoretic heterogeneity in a set of selected proteins is used to estimate the average error frequency during translation. Estimates based upon streptomycin-induced heterogeneity as well as mistranslation of an ochre codon yield an average error frequency of 4 x 10(-4) for normally growing cells.

Escherichia coli↗
PubMed1981

On the accuracy of protein synthesis in Drosophila melanogaster.

We have investigated the accuracy of protein synthesis in somatic tissue of young and senescent Drosophila melanogaster by means of two-dimensional electrophoresis. Electrophoretic heterogeneity characteristic of translational error could not be detected at either age, placing the error frequency below about 4 X 10(-4) per codon. As a positive control on the efficacy of the technique, we showed that electrophoretic heterogeneity is detectable in bacterial cells grown in streptomycin, and that the error frequency calculated from these data is in good agreement with independent measurements. Such cells can tolerate an error frequency of about 3 x 10(-3) without cell lethality, or error catastrophe. Since the Drosophila somatic error frequency is at least eight times lower, even in senescent organisms, translational error is unlikely to be a cause of senescence in Drosophila.

Aging↗
PubMed1980

Postoperative vomiting treated with domperidone. A double-blind comparison with metoclopramide and a placebo.

In a double-blind comparison of domperidone 10 mg, domperidone 4 mg, metoclopramide 10 mg and a placebo, 176 adult patients were given an intravenous injection of one of these substances after postoperative vomiting had occurred. Patients were then observed for 6 hours. Those who failed to respond sufficiently to the double-blind injection were given an open dose of domperidone 4 mg i.v. Vomiting recurred in 52% of patients in each of domperidone groups compared with 75% of patients in the metoclopramide group and 84% of patients in the placebo group. Thus domperidone was significantly more effective than the other two substances (p < 0.05). Significantly more patients in the placebo group required a supplementary injection than in the domperidone and metoclopramide groups (p < 0.05). No side effects were observed in any patient.

Adult↗
PubMed1979

Modern indications for cesarean section.

Total cesarean section rates and rates of various indications for primary cesarean section were reviewed for four yearly periods during the past decade. The time periods studied were chosen to follow the introduction of new obstetric practices and technologic advances in monitoring fetal condition. The total cesarean section rate increased from 6.8% to 17.1% during this time. The most common indication for primary cesarean section was cephalopelvic disproportion, which represented approximately 40% of cases during each study period. Primary cesarean section for fetal distress increased to 28.2% but has decreased over the past 2 years to a present rate of 11.7%. Primary cesarean section for breech presentation increased from 12.3% to 21%. From the perspective of this review a total cesarean section rate of 15% is predicted for the future.

Cesarean Section↗
PubMed1979

A new nucleotide involved in the stringent response in Escherichia coli. Guanosine 5'-diphosphate-3'-monophosphate.

A novel nucleotide has been detected in Escherichia coli subjected to the stringent response. However, this nucleotide does not accumulate in relA+ cells subjected to heat shock, in which guanosine 5'-diphosphate-3'-diphosphate does accumulate but stable RNA synthesis is not restricted. The intracellular level of this new nucleotide thus correlates well with control of stable RNA synthesis. Chemical and enzymatic analysis shows that the new nucleotide is guanosine 5'-diphosphate-3'-monophosphate. It is suggested that this nucleotide may play a role in stringent control of stable RNA synthesis.

Escherichia coli↗
PubMed1979

Error propagation in viable cells.

Error propagation is the process, predicted by theoretical models, whereby errors in translating the genetic code will beget fresh errors in successive generations. It has been postulated that error propagation may underly the mortality of cells which display clonal senescence. We have demonstrated the occurrence of error propagation in viable cells of E. coli during growth in a low concentration of streptomycin, a drug which promotes ribosomal ambiguity. We monitored error propagation by measuring mistranslation of a specific UAA codon, and measured viability by direct enumeration of both live and dead cells through a sensitive microscopic technique. We find that the error frequency may be artificially increased by at least an order of magnitude without generating any detectable increase in the proportion of dead cells or of cells whose descendents are doomed to clonal senescence. The error frequency increases gradually over the course of a few generations, in qualitative agreement with the notion of error propagation, and eventually stabilizes at a constant value much higher than normal. The kinetics of this increase agree quantitatively with the Hoffman-Kirkwood and Holliday formulation of error propagation, for parameter values which dictate convergence to a stable error frequency. This convergent behaviour, under conditions of enhanced mistranslation, demonstrates that the normal parameters are well removed from the region of instability in error propagation; even an order of magnitude increase in mistranslation does not tip the translation system into the unstable mode which has been postulated to underly cell senescence. Thus, the error catastrophe theory of cell senescence cannot apply to the translation system of bacteria. We have reviewed experimental data on the fidelity of translation in somatic cells of higher organisms which militate against the notion that the translation system in these cell types could be much closer to the region of instability than in bacteria. These considerations controvert the error catastrophe theory of cell senescence.

Alkaline Phosphatase↗
PubMed1979

Regulatory nucleotides involved in the Rel function of Bacillus subtilis.

We have examined the accumulation of polyphosphorylated nucleotides in Bacillus subtilis in relation to the function of the rel gene. Our results are as follows. (i) During inhibition of isoleucine activation by O-methylthreonine, wildtype B. subtilis cells accumulate unusual nucleotides with the chromatographic and chemical properties of pppApp, ppApp, pppGpp, ppGpp, pGpp, and ppGp. (ii) During the carbon source downshift elicited by inhibiting glucose uptake, we observed accumulation of the polyphosphorylated guanosine but not adenosine nucleotides. (iii) At the end of long phase in sporulation medium, we observed a small transient accumulation of the polyphosphorylated guanosine but not adenosine nucleotides. (iv) We were unable to detect a nucleotide with chromatographic behavior expected for pppAppp under any conditions. (v) The rel mutant of Swanton and Edlin (Biochem. Biophys. Res. Commun. 46-583-588, 1972) did not accumulate any of these polyphosphorylated nucleotides under any of the conditions examined. (vi) the rel mutant is unimpaired in sporulation. We conclude that one or more of the nucleotides we have detected may be involved in controlling the specificity of transcription during the stringent response, but none of them are required for sporogenesis.

Adenine Nucleotides↗