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Biomedical subjects

A Favre

Publications and source records attributed to A Favre.

At least 109 records · Page 6Linked to original sources
PubMed1987

Partial tRNA deacylation specifically triggers Escherichia coli cell volume reduction.

Limitation of Escherichia coli cell growth rate either by means of continuous 366 nm illumination, which is known to decrease the in vivo acylation level of some tRNA species, or by means of specific inhibitors of tRNA acylation allows the division rate to remain unchanged for a few generations, resulting in cell volume reduction. In contrast the cell volume remains stable or increases after treatment with inhibitors of DNA replication and transcription, or with drugs acting at any other step of protein synthesis. The conclusion that limiting acylation of some tRNA species is the triggering event is confirmed by the use of thermosensitive mutants of aminoacyl-tRNA synthetases or of tRNA (the divE strain mutated in the tRNA1Ser gene). Other cellular responses modulate the expression of cell volume reduction. The relA+ stringent response helps expression of the effect but does not appear to be strictly required. However, cell volume reduction may be masked under conditions triggering the SOS response. The data suggest that tRNA acylation is one of the major steps where cells sense change in their nutrient environment.

Acylation↗
PubMed1986

4-Thiouridine photosensitized RNA-protein crosslinking in mammalian cells.

Monkey kidney cells (CV-1) cultivated in the presence of 0.1 mM 4-thiouridine (S4U) and subsequently illuminated at 365 nm exhibit a marked RNA synthesis inhibition. Maximal effect (approximately 40%) was obtained for a 4 h S4U incubation and a 45 KJ/m2 dose. Under these conditions up to 20% of total cellular RNA is retained at the interphase during phenol-chloroform extraction. The fraction of RNA crosslinked to proteins amounts to 50% of the 3H-uridine labeled RNA synthesized during S4U incorporation and less than 10% for the control samples. This strongly suggests that S4U incorporated within the RNA chains acts as a photoaffinity probe. The data above provide the basis of a method for studying in vivo RNA-protein interactions under non destructive conditions.

Animals↗
PubMed1986

Substitution of uridine in vivo by the intrinsic photoactivable probe 4-thiouridine in Escherichia coli RNA. Its use for E. coli ribosome structural analysis.

In vivo incorporation of the uridine-photoactivable analogue, 4-thiouridine, into the ribosomal RNA of an Escherichia coli pyrD strain has been demonstrated. It is highly dependent on the exogenous uridine and 4-thiouridine concentrations as well as on temperature. We have defined conditions allowing the substitution of 13 +/- 2% of the uridine residues in bulk RNA by 4-thiouridine. On a high-Mg2+ sucrose gradient, 33 +/- 3% of ribonucleic particles sediment as 70S ribosomes, the remaining being in the form of non-associated 50S and 30S particles containing immature rRNA. The thiolated 70S ribosomes tolerate a 4-5% substitution level (40 thiouridine molecules/particle). Surprisingly, 3-4% of ribosomal proteins, about two protein molecules/particle, were spontaneously covalently bound to 4-thiouridine-substituted rRNA. Specific 366-nm photoactivation increased this proportion to 10-12%, i.e. up to six or seven ribosomal protein molecules/particle. The photochemical cross-linking proceeds with apparent first-order kinetics with a quantum yield close to 5 X 10(-3). Although extensive photodynamic breakage of rRNA occurs under aerobic conditions, both the kinetics and yield of ribosomal protein cross-linking were independent of oxygenation conditions. The thiolated (4.5%) 70S ribosomes allowed the poly(U)-directed poly(Phe)synthesis at 48% the control rate. Photoactivation decreased this activity to 28% and 10% when performed under nitrogen and in aerated conditions, respectively.

Escherichia coli↗
PubMed1986

Identification of form III conformers in tRNAPhe from Escherichia coli by intramolecular photo-cross-linking.

In the absence of divalent cations, at neutral pH, low ionic strength, and low to moderate temperature, tRNAs are known to be in a denatured form, designated form III in the tRNA phase diagram by Cole et al. [Cole, P. E., Yang, S. R., & Crothers, D. M. (1972) Biochemistry 11, 4358-4368]. Form III tRNAPhe from Escherichia coli has been studied at pH 7, 5 mM Na+, and 10 degrees C. As judged from ethidium bromide intercalation, it exhibits extensive secondary structure. 4-Thiouridine in position 8 of the tRNAPhe sequence was used as a built-in photoaffinity probe. Spectroscopic and spectrofluorometric analysis in the near-UV range of form III tRNAPhe irradiated with broad-band near-UV light to completion of the reaction before or after reduction with NaBH4 revealed that the Pdo(4-5)Cyt (8-C) and Pdo(4-5)Urd (8-U) adducts form in equimolar yield. In different experiments, the overall yield of s4U conversion to these adducts varies between 20 and 40%. The remaining s4U is photolyzed to weakly absorbing product(s) in the near-UV range. The disappearance of s4U follows biexponential kinetics while the 8-C adduct formation follows monoexponential kinetics, indicating the presence of at least two tRNA classes of conformers, not in equilibrium on the time scale of the reaction. Migration on a denaturing polyacrylamide gel of irradiated form III tRNAPhe revealed three main bands, D1, D2, and D3, and no slowly migrating tRNA dimers. D1 migrates at the control position and presumably contains the photolysis product(s) P. The fast-migrating D2 and D3 bands contain 8-Pyr cross-links which were identified by sequence analysis as 8-(66-68) in D2 and 8-(40-43) and 8-(59-62) in D3. On the basis of these data, it is proposed that the minor poorly photoreactive class II conformers are the cloverleaf and close variants, whereas the major class I cross-linkable conformers are essentially long-extended secondary structures. Clearly, our data demonstrate the polymorphism of form III tRNAPhe.

Base Sequence↗
PubMed1986

Multiple crosslinks of proteins S7, S9, S13 to domains 3 and 4 of 16S RNA in the 30S particle.

Functionally active 70S ribosomes containing 4-thiouracil in place of uracil (substitution level 2%) were prepared by an in vivo substitution method. RNA-protein crosslinks were introduced by 366 nm photoactivation of 4-thiouracil in the purified 30S subunits. Seven single stranded M13 probes containing rDNA inserts complementary to domains 3 and 4 of 16S RNA were constructed. These inserts approximately 100 nucleotides long starting at nucleotide 868 and ending at the 3' OH terminus were used to select contiguous RNA sections. The proteins covalently linked to each selected RNA section were identified by 2D gel electrophoresis. Proteins S7, S9, S13 were shown to be efficiently crosslinked to multiple sites belonging to both domains.

DNA Restriction Enzymes↗
PubMed1986

Near ultraviolet DNA damage induces the SOS responses in Escherichia coli.

The influence of the growth delay induced by near u.v. radiation on the SOS response was monitored by comparing the level of sfiA expression by means of a sfiA::lacZ fusion in both a nuvA+ cell and an isogenic nuvA mutant. The mutant lacks 4-thiouridine in its tRNA and does not exhibit the near u.v.-induced growth delay. Although the two strains exhibit similar sfiA induction levels after 254 nm irradiation, their behaviour is different after illumination with near u.v. light, including solar u.v. Inducibility is 10-20 times higher in the nuvA mutant than in the parent strain. Furthermore, pre-illumination with broad band near u.v. light does not affect the 254 nm-induced sfiA response in the mutant but reduces it by a factor of 3-4 in the parent strain. The kinetics of sfiA induction in near u.v.-illuminated nuvA+ cells, whether treated with 254 nm light or not, is unusual and follows the growth curve: only after 50 min is sfiA derepression observed. It can be concluded that (i) near u.v.-induced DNA lesions are able to trigger the SOS response and (ii) the growth delay effect reduces this response, whether triggered by u.v. or near u.v. light. Hence 4-thiouridine in tRNA acts as a built-in antiphotomutagenic 'device' protecting Escherichia coli cells against mutagenesis and the induction of the SOS response by near u.v. light and sunlight.

DNA Repair↗
PubMed1986

Photosensitized UVA light induction of the SOS response in Escherichia coli.

Several of the factors controlling the extent of the ultraviolet light (and particularly of UVA 320 nm less than lambda less than 380 nm) induced SOS response in E. coli have been studied using a sfiA::lacZ fusion. The decreased 254 nm induced sfiA expression level triggered by a UVA-induced growth delay (Caldeira de Araujo A. & Favre A. (1986) Embo J., 5, 175-179), is closely mimicked by a transient chloramphenicol protein synthesis inhibition. In a nuvA mutant strain (lacking the growth delay effect), UVA light triggers a 30-40% lower SOS response at temperatures higher than 20 degrees C when illumination is performed under anaerobic conditions: endogenous oxygen-mediated photosensitized reactions appear to contribute to the SOS response. In contrast to the temperature independence of the sfiA induction levels obtained after 254 nm irradiation, the UVA induced response is 30-60% lower when the temperature (T) increases from a value lower than 10 degrees C to a value higher than 20 degrees C. This indicates that detoxifying enzymes play a role at T greater than 20 degrees C. Also the in vitro photooxydation of NADH to give NAD+ is described and its possible role in endogenous photosensibilizations discussed. To explain the contrasted mutagenic efficiencies of UVA light treatment when applied to cells in buffer at high fluences, and to growing cells at low fluence rates, we propose that intrinsically the UVA-induced DNA damages are able to trigger the SOS response (cyclobutyl pyrimidine dimers and some O2-dependent lesions) but also constitute premutagenic sites (some lesions leading to alkali-labile DNA breaks).(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1986

An interim assessment of the experience of fast neutron boost in inoperable rectal carcinomas in Orléans.

Since January 1981, 58 patients with postoperative recurrences or inoperable rectal cancer have been included in a trial featuring a neutron boost after an initial series of irradiation given by the classical high energy photon procedure. This group irradiated at the Neutrontherapy Unit in Orléans was compared to a historical series of 52 patients treated by X-rays only at the Tumor Treatment Center of the Hôpital Tenon in Paris. The immediate local control rate for the neutron group was higher than that obtained by irradiation with photon alone (23/31 vs 14/50). Immediate tolerance was generally satisfactory in both groups. The two survival curves are practically identical.

Adenocarcinoma↗
PubMed1985

Induction of size reduction in Escherichia coli by near-ultraviolet light.

Escherichia coli AB1157 cells, growing exponentially at 37 degrees C in 63B1 medium (supplemented with glucose and casamino acids) with a doubling time of 50 min, were subjected to continuous illumination with 366-nm light at a fluence of 1.5 kJ . m-2 X min-1. Under these conditions, the growth rate decreased and after 1 h of illumination, a new stable exponential mode was reached with a doubling-time of 73 min. This reduction in growth rate occurred without any change in the rate of cell division for two generations after the beginning of illumination. Survival was unaffected, implying that cell size must have decreased. This was confirmed with size distribution curves of control and illuminated cells obtained with a Coulter counter. Furthermore electron micrographs of negatively stained cells indicated that illumination results in a 30-40% decrease in cell length, the diameter increasing by 8%. Hence 366-nm light uncouples growth and division rates. Illumination under the above conditions triggered the accumulation in vivo of 8-13-linked tRNA. The stationary level of the 8-13 link, 80% of the maximal level, was reached precisely when the growth rate reached its new stable value. Furthermore, no reduction in growth rate occurred in a nuv- cell lacking 4-thiouridine in its tRNAs. Hence we conclude that the 366-nm photons trigger partial tRNA inactivation with consequent slowing down of protein synthesis and accordingly of the cell growth rate. In addition, the stringent response has at most a minor effect. In conclusion, near-ultraviolet light is able to decrease the rate of cell growth by restricting the availability of charged tRNAs, and this occurs without affecting the cell division rate.

Bacterial Proteins↗
PubMed1984

Role of the stringent response in the expression and mechanism of near-ultraviolet induced growth delay.

The near-ultraviolet (300-400 nm) induced growth delay of Escherichia coli cells was compared in isogenic relA+ and relA- cells illuminated either in the stationary or the exponential phase. In the latter case: (a) the relA- strains of K12 and B/r exhibited similar maximal growth lags (65 min and 55 min respectively); (b) the maximal lags were 1.5-fold and 4-fold longer, respectively, in the isogenic relA+ strains; (c) the rate of the relA- -dependent guanosine 3',5'-bis(diphosphate) (ppGpp) accumulation was three-times lower in the K12 relA+ strain as compared to the B/r relA- strain: (d) a K12 spoT mutant having an impaired rate of ppGpp degradation had a 2-fold longer lag. On the other hand, when illumination is performed in the stationary phase, isogenic relA+ and relA- cells (B/r or K12) exhibited similar growth lags at any fluences, indicating little if any involvement of the stringent response. These data extend previous observations of T.V. Ramabhadran an J. Jagger [(1976) Proc. Natl Acad. Sci. USA, 73, 59-63] but do not support their conclusion that the stringent response is the main factor responsible for growth delay. By monitoring the intracellular level of ppGpp in relA+ spoT- and relA+ spoT+ growing cells during illumination and the subsequent growth lag we observed that the initial burst of ppGpp decreases slowly all along the lag; in all relA+ strains checked the return of ppGpp to its basal level coincides with the recovery of normal growth. We conclude that it is the accumulation of ppGpp over the basal level due either to the stringent response or to prevention of ppGpp degradation that is responsible for an amplification of the growth lag.

Escherichia coli↗
PubMed1984

Metabolism of tRNA in near-ultraviolet-illuminated Escherichia coli. The tRNA repair hypothesis.

The relA+-dependent stringent response is an important component of the mechanism of the near-ultraviolet-induced growth delay. However, the behaviour of the intracellular level of ppGpp is unexpected [Thomas et al. (1981) Eur. J. Biochem. 118, 381-387] and this led us to examine the metabolism of tRNAs during the illumination period and the growth lag that follows. Analysis of the gel electrophoresis migration profiles of tRNA molecules, synthesized prior to the illumination period, provides no evidence for tRNA degradation. Rather, it is suggestive of the rearrangement of some cross-linked tRNA species during the growth lag. By the same technique the neosynthesis of one or several tRNA species escaping the stringent response could be ruled out at the beginning of the growth lag. The behaviour of the cross-linked tRNAs was followed by a chromatographic procedure allowing the quantitative evaluation of the 8-13 link present in vivo. Upon illumination of growing cells, one observes an initial linear increase of the 8-13 link content. Unexpectedly this is followed during the illumination period by an abrupt decrease. The 8-13 link content then remains stable. The data above suggest that part of the 8-13 link (25-40%) is eliminated from tRNA without degradation of the molecules involved. A tRNA repair hypothesis is proposed: elimination of the 8-13 link would occur by scission of the N1-C1' glycosidic bonds at positions 8 and 13 of tRNA. It would be followed by reinsertion of uracil and cytosine in their respective positions.

Binding Sites↗