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C Portier

Publications and source records attributed to C Portier.

At least 19 recordsLinked to original sources
PubMed1992

E.coli polynucleotide phosphorylase expression is autoregulated through an RNase III-dependent mechanism.

It has been previously shown that the pnp messenger RNAs are cleaved by RNase III at the 5' end and that these cleavages induce a rapid decay of these messengers. A translational fusion between pnp and lacZ was introduced into the chromosome of a delta lac strain to study the expression of pnp. In the presence of increased cellular concentrations of polynucleotide phosphorylase, the level of the hybrid beta-galactosidase is repressed, whereas the synthesis rate of the corresponding message is not significantly affected. In the absence of pnp, the level of the hybrid protein increases strongly. Thus, polynucleotide phosphorylase is post-transcriptionally autocontrolled. However, autocontrol is totally abolished in strains where the RNase III site on the pnp message has been deleted or in strains devoid of RNase III. These results suggest that polynucleotide phosphorylase requires RNase III cleavages to autoregulate the translation of its message. Other mutations in the ribosome binding site region support the hypothesis that this 3' to 5' processive enzyme could recognize a specific repressor binding site at the 5' end of pnp mRNA. Implications of these results on the mechanism of regulation and on messenger degradation are discussed.

Base Sequence↗
PubMed1992

Risk assessment in immunotoxicology. I. Sensitivity and predictability of immune tests.

We have previously reported on the design and content of a screening battery involving a "tier" approach for detecting potential immunotoxic compounds in mice (Luster et al., 1988, Fundam. Appl. Toxicol. 10, 2-19). This battery has now been utilized to examine a variety of compounds by the NIEHS Immunotoxicology Laboratory, the National Toxicology Program-sponsored laboratories, and by the Cell Biology Department at the Chemical Industry Institute of Toxicology. The database generated from these studies, which consists of over 50 selected compounds, has been collected and analyzed in an attempt to improve future testing strategies and provide information to aid in quantitative risk assessment for immunotoxicity. Studies presented here have established the ability of each of the tests or test combinations in the screening battery to detect immunotoxic compounds. Efforts are currently underway using this database to determine the relationships between these immune tests and susceptibility to challenge with infectious agents or transplantable tumor cells. The present analyses indicated that the performance of only two or three immune tests are sufficient to predict immunotoxic compounds in rodents (greater than 90% concordance). The tests that showed the highest association with immunotoxicity were the splenic antibody plaque forming cell response (78%) and cell surface marker analysis (83%). The relationship between immunotoxicity and carcinogenicity, as well as genotoxicity, was also determined. These analyses suggested that potential immunotoxic compounds are likely to be rodent carcinogens (p = 0.019) although for compounds that are not immunotoxic the carcinogenic status is unclear. There was no relationship observed between immunotoxicity and mutagenicity as determined using in vitro genotoxicity tests. The significance of these observations is discussed in terms of the relationship between immunotoxicity tests and biological/toxicological processes concerned with human health (e.g., infectious disease).

Animals↗
PubMed1992

Qualitative and quantitative experimental models to aid in risk assessment for immunotoxicology.

We have previously reported on the design and content of a screening battery using a "tier" approach for detecting potential immunosuppressive compounds in mice [1]. This battery was composed of various immune function, immunopathology and host resistance tests, the results of which could help establish the potential of chemical and biological agents to cause immunosuppression. The data from these studies, which now encompass over 50 compounds, have been analyzed in an attempt to improve future testing strategies and provide information to aid in the risk assessment process. Specifically, the following two issues will be addressed; what are the likelihood(s) for each of the individual tests and testing configurations to accurately identify immunotoxic compounds? and what are the quantitative and qualitative relationships between the immune tests and host resistance assays?

Animals↗
PubMed1991

Different specificities of ribonuclease II and polynucleotide phosphorylase in 3'mRNA decay.

We review recent evidence on the in vivo and in vitro mRNA degradation properties of 2 3'-exonucleases, ribonuclease II and polynucleotide phosphorylase. Although secondary structures in the RNA can act as protective barriers against 3' exonucleolytic degradation, it appears that this effect depends on the stability of these structures. The fact that RNase II is more sensitive to RNA secondary structure than PNPase, could account for some differences observed in messenger degradation by the 2 enzymes in vivo. Terminator stem-loop structures are often very stable and 3' exonucleolytic degradation proceeds only after they have been eliminated by an endonucleolytic cleavage. Other secondary structures preceding terminator stem-loop seem to contribute to mRNA stability against exonucleolytic decay.

Base Sequence↗
PubMed1990

Translational control of ribosomal protein S15.

The expression of ribosomal protein S15 is shown to be translationally and negatively autocontrolled using a fusion within a reporter gene. Isolation and characterization of several deregulated mutants indicate that the regulatory site (the translational operator site) overlaps the ribosome loading site of the S15 messenger. In this region, three domains, each exhibiting a stem-loop structure, were determined using chemical and enzymatic probes. The most downstream hairpin carries the Shine-Dalgarno sequence and the initiation codon. Genetic and structural data derived from mutants constructed by site-directed mutagenesis show that the operator is a dynamic structure, two domains of which can form a pseudoknot. Binding of S15 to these two domains suggests that the pseudoknot could be stabilized by S15. A model is presented in which two alternative structures would explain the molecular basis of the S15 autocontrol.

Base Sequence↗
PubMed1990

Translational autocontrol of the Escherichia coli ribosomal protein S15.

When rpsO, the gene encoding the ribosomal protein S15 in Escherichia coli, is carried by a multicopy plasmid, the mRNA synthesis rate of S15 increases with the gene dosage but the rate of synthesis of S15 does not rise. A translational fusion between S15 and beta-galactosidase was introduced on the chromosome in a delta lac strain and the expression of beta-galactosidase studied under different conditions. The presence of S15 in trans represses the beta-galactosidase level five- to sixfold, while the synthesis rate of the S15-beta-galactosidase mRNA decreases by only 30 to 50%. These data indicate that S15 is subject to autogenous translational control. Derepressed mutants were isolated and sequenced. All the point mutations map in the second codon of S15, suggesting a location for the operator site that is very near to the translation initiation codon. However, the creation of deletion mutations shows that the operator extends into the 5' non-coding part of the message, thus overlapping the ribosome loading site.

Base Composition↗
PubMed1990

Target site of Escherichia coli ribosomal protein S15 on its messenger RNA. Conformation and interaction with the protein.

The regulatory site of ribosomal protein S15 has been located in the 5' non-coding region of the messenger, overlapping with the ribosome loading site. The conformation of an in vitro synthesized mRNA fragment, covering the 105 nucleotides upstream from the initiation codon and the four first codons of protein S15, has been monitored using chemical probes and RNase V1. Our results show that the RNA is organized into three domains. Domains I and II, located in the 5' part of the mRNA transcript, are folded into stable stem-loop structures. The 3'-terminal domain (III), which contains the Shine-Dalgarno sequence and the AUG initiation codon, appears to adopt alternative conformations. One of them corresponds to a rather unstable stem-loop structure in which the Shine-Dalgarno sequence is paired. An alternative potential structure involves a "pseudo-knot" interaction between bases of this domain and bases in the loop of domain II. The conformation of several RNA variants has also been investigated. The deletion of the 5'-proximal stem-loop structure (domain I), which has no effect on the regulation, does not perturb the conformation of the two other domains. The deletion of domain II, leading to a loss of regulatory control, prevents the formation of the potential helix involved in the pseudo-knot structure and results in a stabilization of the alternative stem-loop structure in domain III. The replacement of another base in domain III involved in pairing in the two alternative structures mentioned above should induce a destabilization of both structures and results in a loss of the translational control. However, the replacement of another base in domain III, which does not abolish the control, results in the loss of the conformational heterogeneity in this domain and yields a stable conformation corresponding to the pseudo-knot structure. Thus, it appears that any mutation that disrupts or alters the formation of the pseudo-knot impairs the regulatory mechanism. Footprinting experiments show that protein S15 is able to bind to the synthesized fragment and provide evidence that the protein triggers the formation of the pseudo-knot conformation. A mechanism can be postulated in which the regulatory protein stabilizes this particular structure, thus impeding ribosome initiation.

Base Composition↗
PubMed1990

Different specificities of ribonuclease II and polynucleotide phosphorylase in 3'mRNA decay.

We review recent evidence on the in vivo and in vitro mRNA degradation properties of 2 3'-exonucleases, ribonuclease II and polynucleotide phosphorylase. Although secondary structures in the RNA can act as protective barriers against 3' exonucleolytic degradation, it appears that this effect depends on the stability of these structures. The fact that RNase II is more sensitive to RNA secondary structure than PNPase, could account for some differences observed in messenger degradation by the 2 enzymes in vivo. Terminator stem-loop structures are often very stable and 3' exonucleolytic degradation proceeds only after they have been eliminated by an endonucleolytic cleavage. Other secondary structures preceding terminator stem-loop seem to contribute to mRNA stability against exonucleolytic decay.

Base Sequence↗
PubMed1987

Nucleotide sequence of the pnp gene of Escherichia coli encoding polynucleotide phosphorylase. Homology of the primary structure of the protein with the RNA-binding domain of ribosomal protein S1.

The pnp gene is located at 69 min on the Escherichia coli chromosome adjacent to the rpsO gene which encodes the ribosomal protein S15. In this paper, we present the sequence of a 3030-nucleotide DNA fragment containing the open reading frames coding for ribosomal protein S15 and polynucleotide phosphorylase. Translation of pnp is initiated by 5'-UUG-3' codon separated by 7 nucleotides from a good ribosome binding site. Codon usage in this gene is typical of highly expressed proteins of E. coli. Some of the transcripts of the pnp gene terminate just after the stem of the terminator t2 visible in the nucleotide sequence. However, a very strong read-through occurs at this site, thus permitting many of the pnp transcripts to extend beyond this transcription terminator. We also describe the primary structure homologies between a 69-amino-acid stretch of polynucleotide phosphorylase and the four homologous stretches of ribosomal protein S1 which form its RNA binding site. The possibility that this 69-amino-acid stretch constitutes the polynucleotide binding domain of polynucleotide phosphorylase is discussed.

Amino Acid Sequence↗
PubMed1987

The first step in the functional inactivation of the Escherichia coli polynucleotide phosphorylase messenger is a ribonuclease III processing at the 5' end.

The transcripts covering pnp, the gene encoding polynucleotide phosphorylase, are processed by ribonuclease III. In this study, it is shown that the steady state level of the pnp mRNA increased 11-fold in a ribonuclease III-deficient strain. The synthesis rate of this messenger is only slightly affected in the mutant strain whereas the half-life, which is 1.5 min in the wild type, is considerably increased to more than 40 min. Moreover, polynucleotide phosphorylase is 10-fold over-expressed in the mutant strain, which shows that unprocessed pnp mRNA is functional. The position of the ribonuclease III-sensitive site suggests that the sequence involved in the stabilization of the pnp mRNA is located at the 5' end of the message and that the RNase III processing triggers the decay of the transcripts downstream. A similar function for ribonuclease III in the processing of the messenger for the beta beta' subunits of RNA polymerase is proposed.

Endoribonucleases↗
PubMed1986

Initiation, attenuation and RNase III processing of transcripts from the Escherichia coli operon encoding ribosomal protein S15 and polynucleotide phosphorylase.

The rpsO gene of Escherichia coli, which encodes ribosomal protein S15 is located at 69 minutes on the chromosome. It is adjacent to the pnp gene, which encodes polynucleotide phosphorylase. The two genes are separated by 249 nucleotides and are transcribed in the same direction. We report here in vivo S1 nuclease mapping and in vitro transcription experiments that demonstrate that rpsO and pnp are cotranscribed from a promoter P1, located 108 nucleotides upstream from rpsO, and that another promoter P2, located between the two genes 158 nucleotides upstream from pnp, also directs the transcription of pnp. Transcription from P1 can either terminate at the terminator t1 identified in vivo and in vitro, 18 nucleotides downstream from rpsO, or transcribe through t1 and into pnp. Comparison of the transcripts synthesized in wild-type and RNase III-deficient strains of E. coli shows that all the P1 readthrough transcripts and P2 transcripts are cleaved by RNase III. Two specific cuts are made by RNase III in a double-stranded structure about 100 nucleotides upstream rpsO. We also found that some transcripts of this operon start 47 nucleotides downstream from rpsO, in the region of t1. No promoter has been identified in this region. This mRNA is attributed to an endonucleolytic cleavage of the polycistronic transcripts and the location of the cut is named M. The order of the transcription signals and of the maturation sites in relation to rpsO and pnp can be summarized as follows: P1, rpsO, t1, M, P2, RNase III-processing sites, pnp. The possible roles of mRNA processing events in the expression of rpsO-pnp operon are discussed.

Endonucleases↗
PubMed1986

[Unruptured aneurysms of the sinus of Valsalva disclosed by syncopal disorders of cardiac excitability. Apropos of 2 cases].

Aneurysms of the sinus of Valsalva are rarely diagnosed before rupture into the cardiac cavities which usually leads to the appearance of a continuous murmur and cardiac failure. In the two cases described, the presenting symptom of the aneurysm was syncope due to cardiac hyperexcitability: ventricular tachycardia in the first and paroxysmal tachyarrhythmia in the second case. The presenting symptoms of unruptured aneurysms of the sinus of Valsalva were analysed. In general, they are: uncontinuous cardiac murmurs: either diastolic murmurs of aortic regurgitation, systolic murmurs of mitral or tricuspid regurgitation, or, as in our first case, of obstruction to right ventricular ejection; arrhythmias: the commonest are conduction defects, which can be syncopal; hyperexcitability (especially ventricular) seems to be very care. Echocardiography is a valuable tool for the diagnosis of sinus of Valsalva aneurysms. The appearances of unruptured aneurysms in our two patients are described. The presence of syncopal cardiac hyperexcitability, possibly associated with one of the preceding auscultatory abnormalities is an indication for echocardiography which may lead to the diagnosis of this condition.

Aged↗
PubMed1986

The impact of litter effects on dose-response modeling in teratology.

The fitting of dose-response models to teratology data involving littermates in order to generate estimates of teratogenic risk is receiving increasing attention as a potential alternative to the "safety-factor" approach to risk estimation. In this paper, we utilize the beta-binomial distribution to introduce varying degrees of intralitter correlation, and, for purposes of illustration, consider a logistic dose-response model that describes the logit of risk as a straight-line function of ln(dose). The biases and (exact and asymptotic) variances of the maximum likelihood estimators of the intercept and slope are studied by simulation as a function of the intralitter correlation structure.

Animals↗
PubMed1984

Expression of the rpsO and pnp genes: structural analysis of a DNA fragment carrying their control regions.

Precise physical mapping of the genes rpsO and pnp coding respectively for ribosomal protein S15 and polynucleotide phosphorylase together with regions involved in the regulation of their expression has been obtained by the analysis of in vitro deletion mutants. The results suggest that each gene has its own promotor, but that there is coexpression of rpsO and pnp. The nucleotide sequence of rpsO and of the beginning of pnp is presented and includes the presumed regulatory regions of these genes. Several features of the sequence support the mapping experiments and are discussed in relation to the expression of the ribosomal and pnp genes.

Chromosome Deletion↗
PubMed1983

Optimal design of the chronic animal bioassay.

Optimal experimental designs for carcinogenicity bioassays conducted for the assessment of risks associated with exposure to environmental chemicals are derived. For our purposes, an optimal experimental design is a design that minimizes the mean-squared error of the maximum likelihood estimate of the virtually safe dose from the Armitage-Doll multistage model and maintains a high power for the detection of increased carcinogenic response. Three- and four-dose designs (including control as one of the doses) are discussed for a variety of dose response patterns. Monte Carlo simulation techniques are used to estimate the power and mean-squared error for small samples sizes. Two forms of the multistage model are used to estimate the virtually safe dose: the linear model and the linear-quadratic model. The optimal designs for fitting the linear model used a control group and a group administered the maximum tolerated dose, with about 50% of the animals at each dose. The three- and four-dose optimal designs when fitting the linear-quadratic model were found to be equivalent. However, after considering several biological issues, including overt toxicity, the optimal four-dose designs would use between 150 and 300 animals, with 50 to 60 animals in the control group, and 40 to 60 animals in the group administered the maximum tolerated dose. One-third of the remaining animals would be administered a dose between 10 and 30% of the maximum tolerated dose, and two-thirds of the remaining animals would be administered 50% of the maximum tolerated dose.

Animals↗
PubMed1983

Low-dose-rate extrapolation using the multistage model.

The distribution of the maximum likelihood estimates of virtually safe levels of exposure to environmental chemicals is derived by using large-sample theory and Monte Carlo simulation according to the Armitage-Doll multistage model. Using historical dose-response we develop a set of 33 two-stage models upon which we base our conclusions. The large-sample distributions of the virtually safe dose are normal for cases in which the multistage-model parameters have nonzero expectation, and are skewed in other cases. The large-sample theory does not provide a good approximation of the distribution observed for small bioassays when Monte Carlo simulation is used. The constrained nature of the multistage-model parameters leads to bimodal distributions for small bioassays. The two modes are the direct result of estimating the linear parameter in the multistage model; the lower mode results from estimating this parameter to be nonzero, and the upper mode from estimating it to be zero. The results of this research emphasize the need for incorporation of the biological theory in the model-selection process.

Biological Assay↗
PubMed1981

Cloning of E. coli pnp gene from an episome.

Starting with an F' episome harboring a transposon inserted in the pnp gene (Portier 1980), we were able to identify an EcoRI restriction fragment carrying the pnp and argG genes. This fragment, from both wild-type and mutant episomes, was cloned ni pACYC184. The presence of argG on the fragment allowed positive selection of the desired clones in an auxotrophic strain (argG). A restriction map was established and a fragment of 3 megadaltons subcloned in the plasmid vector pBR322. The pnp gene corresponds to about 50% of this subcloned segment and was roughly located by deletion mapping. The direction of transcription and locations of the promotor and gene extremities were determined by analyzing proteins synthesized in "maxi-cells". In addition, the gene coding for a 10,000 dalton protein was found to reside adjacent to the beginning of the pnp structural gene. Strains carrying plasmids which express the pnp overproduce polynucleotide phosphorylase.

Arginine↗