Biomedical subjects

C Reiss

Publications and source records attributed to C Reiss.

At least 19 recordsLinked to original sources

PubMed2000

Conformational toxicity and sporadic conformational diseases.

Spontaneous, so-called 'conformational' diseases, specially of the neurodegenerative type like Alzheimer's, are linked to certain protein types which have the normal amino-acid sequence but are misfolded and accumulate due to resistance to proteolysis. In the case of prion diseases, the 'protein only' hypothesis assumes that the misconformation of a native protein could be initiated upon interaction with a sister-protein already in the misfolded state. There is an alternative to this sister protein contamination scheme, which assumes that the misconformation is acquired upon protein synthesis, that is de novo. Misfoldling and resistance to proteolysis could result from defects responsible for shortage or inactivity of the cellular factors in charge of protein folding and degradation. The defects could have a genetic origin (the gene of the faulty factor involved could have been mutated, or control and regulation of its expression could have been altered, etc.). Alternatively, the cell's actual biosynthetic and/or proteolytic resources could have become overloaded and unavailable, due to unscheduled mass-production of proteins resulting from unscheduled cell growth or proliferation, cell stress, etc. Xenobiotics, active for instance as endocrine proliferators, stressors, or inducing copious, unscheduled gene expression, etc. could give rise to shortage of cellular factors necessary for the production of native proteins and for proteolysis. Alternatively, xenobiotics could alter expression or activity of some of these factors. In both cases, the xenobiotic could be a 'conformational toxicant' by inducing misfolding of selected proteins. The xenobiotic could trigger some conformational disease if it targets a specific protein and tissue.

Animals↗

PubMed2000

Ribosome traffic in E. coli and regulation of gene expression.

The ribosome traffic during translation of E. coli coding sequences was simulated, assuming that the rate of translation of individual codons is limited by the cognate tRNA availability. Actual translation rates were taken from Solomovici et al. (J. theor. Biol. 185, 511-521, 1997). The mean translation rates of the 4271 sequences cover a broad, two-fold range, whereas the local rate of translation along messengers varies three-fold on average. The simulation allows one to sketch the ribosome traffic on the polysome, in particular by providing the extent of mRNA sequences uncovered between consecutive ribosomes and the time during which these sequences are exposed. These parameters may participate in the control of mRNA stability and transcriptional polarity. By averaging the translation rates in a 17-codon window, assumed to be the sequence covered by a translating ribosome, and sliding this window along a given coding sequence, the addresses KMAX and KMIN, and the times TMAX and TMIN of respectively the slowest and the fastest translated window were determined. It is shown that under the assumptions made, TMAX sets the number of proteins translated from a given mRNA molecule per unit time, in case the delay between consecutive translation starts is below TMAX. Both windows display two strong biases, one as expected on the usage of codon frequencies, and the other surprisingly on the occurrence of amino acids.

Amino Acids↗

PubMed1999

Synonymous codon substitutions affect ribosome traffic and protein folding during in vitro translation.

To investigate the possible influence of the local rates of translation on protein folding, 16 consecutive rare (in Escherichia coli) codons in the chloramphenicol acetyltransferase (CAT) gene have been replaced by frequent ones. Site-directed silent mutagenesis reduced the pauses in translation of CAT in E. coli S30 extract cell-free system and led to the acceleration of the overall rate of CAT protein synthesis. At the same time, the silently mutated protein (with unaltered protein sequence) synthesized in the E. coli S30 extract system was shown to possess 20% lower specific activity. The data suggest that kinetics of protein translation can affect the in vivo protein-folding pathway, leading to increased levels of protein misfolding.

Amino Acid Sequence↗

PubMed1998

Silent mutations in the Escherichia coli ompA leader peptide region strongly affect transcription and translation in vivo.

In order to test the effect of silent mutations on the regulation of gene expression, we monitored several steps of transcription and translation of the ompA gene in vivo , in which some or all codons between codons 6 and 14, frequently used in Escherichia coli , had been exchanged for infrequent synonymous codons. Northern blot analysis revealed an up to 4-fold reduction in the half-life of the mutated messengers and a >10-fold reduction in their steady-state amounts. Western blot analysis showed a 10-fold reduction in the amount of OmpA protein. Use of a system expressing a Rho-specific anti-terminator allowed us to detect a strong transcription polarity effect in the silent mutants. These results demonstrate that silent mutations can severely inhibit several steps of gene expression in E. coli and that code degeneracy is efficiently exploited in this species for setting signals for gene control and regulation.

Bacterial Outer Membrane Proteins↗

PubMed1998

[3H]MDL 105,519 binds with equal high affinity to both assembled and unassembled NR1 subunits of the NMDA receptor.

[3H]MDL 105,519 (((E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1[3H]-indole-2-ca rboxylic acid) is a novel radioligand which binds with high affinity, Kd = 2.5 nM, to the glycine site of adult rodent forebrain, N-methyl-D-aspartate subtype of glutamate receptors. As with other glycine site antagonists, the major determinants for high-affinity binding of [3H]MDL 105,519 resides upon the NRI subunit, and not the NR2 subunits. [3H]MDL 105,519 binds with equal affinity, Kd = 3 nM, to both NR1-1a or NR1-4b splice variants, as well as the NRI-1a/NR2A receptor expressed in human embryonic kidney (HEK) 293 cells. One percent Triton X-100/1 M NaCl solubilises with a recovery of 15+/-3%, a mixed pool of assembled and unassembled forebrain NR1 subunit polypeptides. In this preparation, the recovery of [3H]MK801 ((+)-5-[3H]methyl-10,11-dihydrodibenzo[alpha,d]cyclohepten-5 ,10-imine binding activity (7+/-1%) reflects the amount of assembled NR1 subunits whereas [3H]MDL 105,519 binds quantitatively, with a recovery of 19+/-4% and Kd = 3 nM, to both assembled and unassembled NRI subunits. Therefore, [3H]MDL 105,519 should prove a useful ligand, in conjunction with immunopurification approaches, to address the question of NMDA receptor subunit stoichiometry.

Animals↗

PubMed1998

Detection of transmembrane helical segments at the nucleotide level in eukaryotic membrane protein genes.

The analysis of base distributions at the three codon positions, in sequences coding for polytopic membrane proteins from Eukaryotes, reveals a global excess of thymine and a depletion of adenine, at the second codon position. These genes were scanned using a sliding window, in which the average ratio of T over A at the second position of codons was computed. The scan shows that sharp peaks of this ratio, which are responsible for the high mean value of this parameter in the genes, correlate closely with the transmembrane segments of the membrane proteins. These results are quite similar to our previous findings for bacterial polytopic inner membrane proteins. This establishes the ratio of T over A at codon position two as a universal parameter for both the characterization of the genes of polytopic membrane proteins, and for the location of alpha-helical, transmembrane segments of these proteins.

Codon↗

PubMed1998

Enhanced expression of the yeast Ure2 protein in Escherichia coli: the effect of synonymous codon substitutions at a selected place in the gene.

The expression of the yeast Ure2 protein and its two N- and C-terminal HA-(YPYPVDYA) epitope and His-tag fusions has been enhanced in E. coli by selected silent mutagenesis of the URE2 gene. The two Arg-AGA codons at positions 253 and 254 of the URE2 gene coding sequence were exchanged by CGT codons accordingly. This has allowed an increased yield (up to 100-fold) of the full-length protein synthesized. Western blotting with HA-epitope-specific antibodies using N- and C-terminal Ure2p-HA(epitope)-His-tag fusion constructs confirmed the integrity of the recombinant proteins. The N-(C-) terminal tagged proteins were shown to possess biological activity of the natural Ure2 protein.

Amino Acid Sequence↗

PubMed1997

Differential resistance to proteinase K digestion of the yeast prion-like (Ure2p) protein synthesized in vitro in wheat germ extract and rabbit reticulocyte lysate cell-free translation systems.

The Ure2p yeast prion-like protein was translated in vitro in the presence of labeled [35S]methionine in either rabbit reticulocyte lysate (RRL) or wheat germ extract (WGE) cell-free systems. When subjected to proteinase K digestion, the Ure2p protein synthesized in WGE was proteolysed much more slowly compared to that synthesized in RRL; this displays fragments of about 31-34 kDa, persisting over 8 min. Thus, the digestion rate and pattern of the protein synthesized in WGE, unlike that synthesized in RRL, revealed characteristic features of the [URE3] prion-like isoform of the Ure2p protein [Masison, D.C. and Wickner, R.B. (1995) Science 270, 93-95]. Chloramphenicol acetyltransferase, synthesized under the same conditions, differed fundamentally in its proteolytic sensitivity toward proteinase K (PK); in the RRL system it was more slowly digested than in WGE, proving specific PK inhibitors to be absent in both systems. Posttranslational addition of the WGE to the RRL-synthesized Ure2p does not protect Ure2p from efficient PK degradation either. The differences in Ure2p degradation may be ascribed to a specific structure or specific states of association of Ure2p synthesized in WGE; obviously, they yield a protein that mimics the behavior of the Ure2p in [URE3] yeast strains. The present data suggest that particular conditions of the Ure2p protein translation and/or certain cellular components (accessory proteins and extrinsic factors), as well as the nature of the translation process itself, could affect the intracellular folding pathway of Ure2p leading to the de novo formation of the prion [URE3] isoform.

Animals↗

PubMed1997

Does Escherichia coli optimize the economics of the translation process?

The codon translation rate is usually assumed to be proportional to the cellular concentration of the cognate tRNA, but synonymous codons sharing the same cognate tRNA may be translated at rather different rates. To account for the latter observation, we assume that the translation process is optimized in two respects: (i), the codon demand is optimized with respect to the supply of cognate tRNAs (composition of the tRNA pool); and (ii), for synonymous codons sharing the same cognate tRNA, the usage frequency of each codon correlates optimally with the stability of the codon-anticodon complex. These assumptions allow us to compute the relative rate constants of synonymous codons. Highly expressed genes, which produce 80-90% of the protein mass in the E. coli cell, appear to have selected codons which make an optimal use of the tRNA pool. Assuming the optimization criteria were valid, a list of codon translation times (in ms) were derived from available experimental data.

Codon, Terminator↗

PubMed1996

Synonymous codon selection controls in vivo turnover and amount of mRNA in Escherichia coli bla and ompA genes.

A number of silent codon changes were made in two Escherichia coli genes. For the ompA gene, the replacement of seven consecutive frequently used codons with synonymous infrequently used codons reduced the ompA mRNA level and its half-life. For the bla gene, the exchange of 24 codons for the most frequently used synonymous codons extended the bla mRNA half-life. A modification of ribosome traffic could account for these observations.

Amino Acid Sequence↗

PubMed1996

A method to detect transmembrane helical segments at the nucleotide level.

The analysis of base distributions at the three codon positions, in sequences coding for integral inner membrane proteins from Bacteria, reveal a global excess of thymine and a depletion of adenine, at codon position two. These genes were scanned using a sliding window, in which the average ratio of T to A at the second position of codons, TA(2) (converted to a logarithm, the LTA2 ratio) was computed. The profiles obtained reveal sharp and local peaks of the LTA2 ratio, which account for the high mean values of this parameter. For inner membrane proteins of known structure, the position and extent of these peaks correlate with the location of transmembrane alpha-helices. The prediction accuracy of the detection of such structures using the LTA2 ratio compares to that obtained using algorithms based on an hydrophobicity index of amino-acids. This new criterion presents several advantages, as it deals directly with the nucleotide sequence, does not rely on the values of empirical parameters and finds direct applications in molecular biology studies. Since the nucleotide sequences of transmembrane beta-strands do not give rise to peaks in the LTA2 ratio profile, the criterion appears to characterize exclusively alpha-helical segments in transmembrane proteins.

Bacteria↗

PubMed1995

Kinetic study in vitro of Escherichia coli promoter closure during transcription initiation.

The rate of closure of two Escherichia coli promoters borne by plasmid pBR322, following transcription initiation from the open complex, was probed in vitro by the protection of unpaired thymines in the open complex against oxidation by KMnO4. Run-off transcription kinetics were also studied under identical conditions. Closure of the open promoter appears to be by far the rate-limiting step of transcription initiation and elongation for the linearized beta-lactamase gene, and is strongly dependent on template topology for the RNAI gene. It is suggested that the corresponding signals are deposited 30 bases at least downstream of transcription initiation and that promoter closure, and its clearance by elongating RNA polymerase, may occur almost simultaneously.

Base Sequence↗

PubMed1993

[Stability of messenger RNA of Escherichia coli ompA is affected by the use of synonymous codon].

The ompA gene of E. coli was silently mutated by the substitution of nine codons located towards the end of the gene, by synonymous codons expected to be translated more slowly. At 37 degrees C in vivo the life-time of the ompA messenger RNA (mRNA) is reduced from 4.5 min (w.t.) to 3.8 min (silent mutant). The amount of mRNA of the silent mutant is only 30% of that observed for the w.t. gene. These variations are thought to be due to the uncoupling of transcription from translation, and a lesser protection of the mRNA towards RNase digestion by ribosomes, resulting from a lesser density of the ribosome traffic on the mutated polysome.

Blotting, Northern↗

PubMed1993

The collision of cotranscribing E. coli RNA polymerases studied in vitro.

The collision of cotranscribing E. coli ternary complexes was brought about in vitro by forcing the first "leading" complex to stall before first template C, due to the absence of CTP, and then permitting initiation of a second "following" complex. Following collision, the transcript of the leading complex was increased in length by as much as 7 nt., despite the absence of CTP, but did not dissociate. Upon addition of CTP, the leading complex aborted at exactly the positions requiring incorporation of the next Cs', the following complex continued transcription. The observations point to the importance of linking appropriate promoter efficiency with transcriptional pausing times, and the role of transcriptional collisions in termination events.

Adenosine Triphosphate↗

PubMed1993

Transcription pausing signal detected by sense/antisense transcription.

We studied elongation pausing during transcription in vitro on both the sense and the antisense strands of a given gene fragment(5' end of the bla gene of Tn3). The average transcription rate on the sense strand was much lower than that of the antisense strand, and several pauses observed on the former have no detectable antisense partner. A pausing signal was identified, associated with an (AT)6 sequence, in the vicinity of a strong pause on the sense strand and the sole detected pause on the antisense strand. Mutation of this sequence strongly reduced, by a common factor, pausing times at both sites.

Base Sequence↗

PubMed1992

In vivo control of promoter and terminator efficiencies at a distance.

In pBR329, the genes providing resistance to ampicillin (beta-lactamase, bla) and chloramphenicol (chloramphenicol acetyl transferase, cat) are encoded on the same strand. The bla gene lies downstream of the cat gene, separated by an intergenic sequence of 414 bp. The transcription starts of the two genes are 1090 bp apart. We have probed, in vivo, the effect on transcription of the bla gene, of the introduction, in front of the cat gene, of a series of synthetic promoters covering a large (over 60-fold) range of efficiency. The rising efficiency of the cat promoter has several important consequences for transcription of the bla gene. First, a strong (up to sevenfold) stimulation of the bla promoter is observed, together with a shift of the main bla transcription start site, 10 bp upstream. Furthermore, the relative efficiencies of the bla transcription terminators are reduced. Finally, because of a lesser relative efficiency of the cat transcription terminators as well, we observe enhanced intrusion into the bla gene of transcripts initiated at the cat promoter, some of them extending to the bla transcription terminator and beyond. The operon-like expression of the cat-bla gene tandem is controlled by the efficiency of the cat terminator, which in turn depends on that of the cat promoter. This demonstrates a direct link between the efficiencies of promoter and terminator. Upon inhibition of bacterial gyrase activity, i.e. relaxation of negative supercoiling action, bla expression increases sharply in pBR329, but remains almost unchanged in a plasmid (pBRGC-1) in which cat is under the control of a 6.5-fold stronger promoter. Therefore, under normal gyrase activity, the stimulation of the bla promoter in pBRGC-1 (relative to pBR329) appears to be linked to topological relaxation of its template in situ, in keeping with earlier in vitro observations. We propose that the relaxed state of pBRGC-1 in situ could be due to the decrease in the plasmid linking number, introduced by the 10-12 RNA polymerases that simultaneously transcribe the cat gene in that plasmid, compared with only one or two in pBR329. We find that the negative superhelical densities of both plasmids are almost identical when extracted from the cell. Therefore gyrase would not correct for the relaxed state of plasmid pBRGC-1 observed in situ.

Ampicillin Resistance↗

PubMed1991

Kinetics of the specific binding of a second RNA polymerase to the standard bacterial-transposon-Tn3 bla promoter complex.

It was shown previously, that at moderate excess of RNA polymerase (RNAP) relative to DNA, the complex of the bla promoter from bacterial transposon Tn3 with RNAP is locked in an inactive, heparin-resistant, isomeric state which is not that of the 'open' complex. This 1:1 isomer can accommodate a second RNAP, which becomes tightly and specifically bound just upstream of the first RNAP [Duval-Valentin & Reiss (1990) Mol. Microbiol. 4, 1465-1475]. Both the resulting 2:1 complex and its antecedent 1:1 complex formed at excess of RNAP are immediately and permanently inhibited for transcription initiation. Using the gel-retardation technique, we investigate here the kinetics of formation and decay of the 2:1 complex under various experimental conditions. The data are consistent with pseudo-first-order kinetics at moderate excess of RNAP. The salt-dependence of rate and equilibrium constants has been analysed within the framework of the theoretical model described by Lohman, Dehaseth & Record [(1978) Biophys. Chem. 8, 281-294]. It was found that the salt-dependence is consistent with the existence of a transient intermediate during formation of the 2:1 complex, which forms rapidly on the time scale of its isomerization to the final 2:1 complex. The intermediate is characterized by the release of about seven cations from the 1:1 complex, one additional cation being released upon its final isomerization. Formation of the 2:1 complex at high excess of RNAP becomes inhibited, probably as a result of a 'bumping' effect of the complex by the enzyme, also observed with several other promoters. We conclude that formation of the 2:1 complex closely mimics that of the standard 1:1 complex, except that the final isomerization step to an 'open' complex is lacking. A mechanism of the formation of the 2:1 complex and of its role in transcription regulation of constitutive promoter by RNAP is proposed.

DNA Transposable Elements↗

PubMed1991

Effect of respiratory syncytial virus infection on mice with protein malnutrition.

Respiratory syncytial virus (RSV) pulmonary infection was produced in BALB/c mice fed protein-deficient diets in an effort to understand the severity of viral pneumonia in infants in developing countries. As in previously published experiments with Sendai virus, animals on the deficient diet became clinically malnourished, and certain aspects of their cell-mediated immunity were altered. The course of RSV infection in protein-deprived mice was essentially identical to that in normally nourished animals. The titer of virus recovered from lung homogenates over time, as well as the histologic picture of bronchiolitis, were identical under all experimental conditions. This model, unlike that of Sendai virus infection, fails to demonstrate an effect of protein malnutrition on RSV infection.

Animals↗