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Biomedical subjects

C Gualerzi

Publications and source records attributed to C Gualerzi.

At least 19 recordsLinked to original sources
PubMed1995

Specific protection of 16 S rRNA by translational initiation factors.

The binding of initiation factors to 30 S ribosomal subunits protects specific sets of nucleotides in 16 S rRNA from base-specific chemical probes. Initiation factor 3 (IF-3) protects residues G700, G703 and G791 from attack by kethoxal. These protected bases are close to those in 16 S rRNA that are protected by 50 S subunits, providing a structural basis for the subunit dissociation activity of IF-3. The IF-3-dependent protections also flank bases that are protected by P-site-bound tRNA, in keeping with the possibility that IF-3 may interact with initiator tRNA, or influence the properties of the 30 S P site during initiation. IF-1 protects G530, A1492 and A1493 and causes enhanced reactivity of A1408. These bases are precisely the ones that are protected by the binding of tRNA to the ribosomal A site. This suggests that IF-1 mimics A-site-bound tRNA, and could serve to prevent premature binding of aminoacyl tRNA by blocking the 30 S A site. We were unable to detect any effect of IF-2 on the reactivity pattern of 16 S rRNA, suggesting that this factor may interact primarily through protein-protein interactions.

Aldehydes↗
PubMed1995

Occurrence of inosine kinase as a distinct enzyme in Spirulina platensis.

Among a series of purine nucleosides, inosine was found to be phosphorylated at the highest rate by crude extracts of the cyanobacterium Spirulina platensis. The inosine phosphorylating activity could be separated from hypoxanthine-guanine phosphoribosyl transferase. This result shows that IMP formation may occur via the direct phosphorylation of inosine at its 5'-position, rather than via inosine phosphorolysis, followed by hypoxanthine phosphoribosylation, and provides unequivocal evidence for the occurrence of inosine kinase in nature.

Cyanobacteria↗
PubMed1991

Mutations altering chromosomal protein H-NS induce mini-Mu transposition.

Bacteriophage Mu is one of the most efficient transposons known, capable of moving a hundred viral copies to new positions in the bacterial chromosome in an hour. Mu also forms stable lysogens. In bacteria lysogenic for the defective protein fusion-forming phage MudII1681, which can transpose and replicate but does not encode genes for DNA packaging and cell lysis, the frequency of transposition changes as colonies age. To find host genes that alter the spontaneous Mu transposition frequency, we used a genetic screen with mini-MudlacZ fusion formation as an assay. H-NS (also called H1a and B1) is an abundant nonspecific DNA-binding protein localized to the bacterial chromosome. H-NS has an unusual structure of interspersed patches of acidic and basic residues reminiscent of eukaryotic HMG proteins. Mutations in hns caused an increase in Mu-specific transcription and a dramatic increase in MudII1681 transposition rates when cells were put under certain growth conditions. Purified H-NS stabilized Mu repressor-DNA complexes in vitro, suggesting that H-NS contributes to the organization of transcriptionally inactive DNA in vivo.

Amino Acid Sequence↗
PubMed1991

Vaginosonography in primary prevention of endometrial oncological pathology.

In 38 patients observed for postmenopausal bleeding endometrial thickness measured by vaginosonography before diagnostic curettage was compared with histological results. In 30 patients with endometrial thickness below 4 mm only inactive endometrium was found, whereas in 8 patients with endometrial thickness above 4 mm different pathologies were present. The role of vaginosonography in primary prevention of endometrial oncology is discussed.

Endometrium↗
PubMed1990

Translational control of prokaryotic gene expression.

Awareness of the importance of post-transcriptional control of gene expression in prokaryotes has grown enormously over the past ten years. In particular, translation features as a step where both control over constitutive rates of gene expression, as well as cis and trans regulation are exercised. Recent research has provided us with new insights into the molecular basis of these phenomena.

Escherichia coli↗
PubMed1989

Ribosomal affinity and translational initiation in Escherichia coli. In vitro investigations using translational initiation regions of differing efficiencies from the atp operon.

The atp operon of Escherichia coli comprises nine genes that are differentially expressed. The control of the atp genes' expression rates has been shown to be exercised primarily at the level of translational initiation, but how is this achieved in molecular terms? In order to study the interactions of 30 S ribosomal subunits with specifically the translational initiation regions (TIRs) of atpB, atpE and atpG, restriction fragments bearing these TIRs were excised from the atp operon and cloned into an SP6 promoter transcription vector. mRNA transcripts were made in vitro and used in primer extension inhibition studies and equilibrium mRNA-30 S ribosomal subunit binding measurements. The binding of 30 S ribosomal subunits blocked primer extension 14 to 15 bases downstream from the respective translational start codons. The affinities of binding of 30 S ribosomal subunits showed the relationship atpE greater than atpB greater than atpG. This was also the order of the efficiency of translation promoted by the respective TIRs, both in vivo and on the in vitro synthesized mRNA fragments. Thus, the affinity of 30 S ribosomal subunits is at least to some extent correlated with the rate of translational initiation.

Base Sequence↗
PubMed1988

Interaction of bovine mitochondrial ribosomes with Escherichia coli initiation factor 3 (IF3).

Mammalian mitochondrial ribosomes are distinguished from their bacterial and eukaryotic-cytoplasmic counterparts, as well as from mitochondrial ribosomes of lower eukaryotes, by their physical and chemical properties and their high protein content. However, they do share more functional homologies with bacterial ribosomes than with cytoplasmic ribosomes. To search for possible homologies between mammalian mitochondrial ribosomes and bacterial ribosomes at the level of initiation factor binding sites, we studied the interaction of Escherichia coli initiation factor 3 (IF3) with bovine mitochondrial ribosomes. Bacterial IF3 was found to bind to the small subunit of bovine mitochondrial ribosomes with an affinity of the same order of magnitude as that for bacterial ribosomes, suggesting that most of the functional groups contributing to the IF3 binding site in bacterial ribosomes are conserved in mitochondrial ribosomes. Increasing ionic strength affects binding to both ribosomes similarly and suggests a large electrostatic contribution to the reaction. Furthermore, bacterial IF3 inhibits the Mg2+-dependent association of mitochondrial ribosomal subunits, suggesting that the bacterial IF3 binds to mitochondrial small subunits in a functional way.

Animals↗
PubMed1986

Proteins from the prokaryotic nucleoid: 1H NMR study of the quaternary structure of Escherichia coli DNA binding protein NS (HU).

The quaternary interactions of Escherichia coli DNA binding proteins NS1, NS2, and NS (NS1 + NS2) have been studied by 1H NMR spectroscopy at 400 MHz following the reversible spectral changes produced by temperature increases on the resonances (Phe ring and His C-2 protons) whose spectral characteristics reflect the formation and dissociation of either homologous or heterologous interactions. These changes include (a) a progressive intensity decrease of the Phe resonances shifted to high field by stacking interactions, (b) a progressive intensity increase of the resonances due to freely rotating Phe, and (c) splitting of the His C-2 proton resonance. The association constants and thermodynamic parameters for the homologous and heterologous interactions were calculated from the molar fractions of the relevant molecular species by assuming that the above effects are due to the existence of simple association equilibria. It was found that two (out of three) phenylalanine residues of each polypeptide chain are involved in quaternary interactions. Quantitative data concerning the internal mobility and mutual orientations in aggregates of these Phe rings were also obtained. From the calculated association constants, from comparison of these data with recent protein-protein cross-linking results [Losso, M. A., Pawlik, R. T., Canonaco, M. A., & Gualerzi, C. O. (1986) Eur. J. Biochem. 155, 27-32], and from other considerations, we suggest that even though stacking of the Phe rings occurs at the interface between monomers, the temperature-dependent alteration of the Phe spectrum monitors shifts of the dimer in equilibrium tetramer equilibrium whereas the splitting of the His C-2 proton resonance most likely monitors the equilibrium between tetramers and larger aggregates.

Bacterial Proteins↗
PubMed1985

Linear diffusion of restriction endonucleases on DNA.

We have investigated the dependence of the rate of cleavage of DNA by EcoRI, HindIII, and BamHI on the chain length of the substrate. In order to keep the influence of flanking sequences and of nonspecific binding identical for all substrates we have carried out all experiments with the same plasmid DNA which had been digested previously with a variety of different restriction enzymes to give a set of substrates of different lengths. Our results show that depending on the buffer conditions long substrates are cleaved faster than small ones. We interpret these findings to mean that under certain conditions a linear diffusion of the enzymes on the DNA is involved in localizing the recognition sites. For EcoRI the mean diffusion length is approximately 1000 base pairs at 1 mM MgC12 which can be shown by diffusion theory to correspond to a linear diffusion coefficient of 5 X 10(-10) cm2 s-1. At 10 mM MgCl2 the linear diffusion of EcoRI is negligible and does not lead to a significant enhancement of the rate of site localization. In the presence of nonsaturating amounts of one of the prokaryotic histone-like protein Hu (NS 2) small and large DNA substrate are cleaved with identical rate by EcoRI indicating that other proteins bound to the DNA constitute a barrier across which linear diffusion cannot take place. We conclude that linear diffusion, albeit detectable under certain conditions in vitro, probably is of little importance for the process of site localization in vivo.

Binding Sites↗
PubMed1985

The interaction between initiation factor 3 and 30 S ribosomal subunits studied by high-resolution 1H NMR spectroscopy.

The interaction between Escherichia coli translational initiation factor 3 (IF-3) (Mr = 20668) and 30 S ribosomal subunits or fragmented 16 S rRNA was followed by 1H NMR spectroscopy. Upon addition of increasing yet largely substoichiometric amounts of deuterated 30 S ribosomal subunits, selective line broadenings and some chemical shift changes were observed. These effects can be fully reversed by increasing the temperature and/or the ionic strength. The selective line broadenings, which are explained by a medium-fast to fast exchange dynamics between free and bound IF-3 with loss of internal mobility of the protons, shed light on the amino acid residues of IF-3 involved in or affected by the binding to the 30 S subunits. Some effects (i.e. implication of 1 tyrosine, 1 phenylalanine, and some arginine and lysine residues) are seen with both 30 S subunits and rRNA while others (i.e. implication of a second tyrosine or phenylalanine residue of a group of hydrophobic residues and, possibly, of the single histidine residue), seen only or preferentially with 30 S subunits, may reflect additional interactions exclusively occurring at the ribosomal level.

Amino Acids↗
PubMed1985

Amniotic fluid thyrotropin (TSH) following maternal administration of thyrotropin releasing hormone.

Cord blood and amniotic fluid thyrotropin (TSH), T4, T3, and rT3 concentrations were measured in 49 women who received 400 micrograms thyrotropin releasing hormone (TRH) iv during labor and in 16 control women who received saline. Cord blood serum TSH concentrations were elevated for as long as 4 hours after TRH administration and peak values (38.0 +/- 4.2 microU/ml) were observed from 61-120 minutes after TSH as compared to control values of 5.0 +/- 0.3 microU/ml. The elevations in fetal TSH concentration stimulated the fetal thyroid, resulting in a progressive increase in cord blood T4 and T3 but not rT3 concentrations. These TRH induced elevations in fetal cord blood TSH concentrations were not accompanied by increases in unconcentrated and 4 fold concentrated amniotic fluid TSH concentrations which were almost always below 0.6 microU/ml, the limit of assay sensitivity. Unconcentrated amniotic fluid T4 concentrations were barely detectable and no variation was observed between the TRH treated and saline treated mothers; amniotic fluid T3 was not detectable in any of the groups; and amniotic fluid rT3 concentrations ranged between 46.4 and 55.6 ng/dl and did not differ between groups. These findings suggest that term amniotic fluid TSH values do not reflect transient but marked elevations in fetal serum TSH concentrations and that amniotic fluid TSH determination is probably not useful in the detection of primary fetal hypothyroidism. It is possible, but unlikely, that long-term and even greater elevations in fetal serum TSH concentrations would result in increased amniotic fluid TSH concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Amniotic Fluid↗
PubMed1984

Effect of polyamines and basic proteins on cleavage of DNA by restriction endonucleases.

We have investigated the effect of the polyamines spermine, spermidine, and putrescine and the prokaryotic histone-like proteins NS1 and NS2 on the restriction endonuclease EcoRI catalyzed cleavage of plasmid and bacteriophage DNAs. At low concentrations of spermine and spermidine, the rate of DNA cleavage by EcoRI is increased, while high concentrations of spermine as well as of spermidine are inhibitory. These phenomena are also observed with other restriction endonucleases. They are, therefore, probably due to the interaction of the polyamines with the DNA. Putrescine does not have such an effect within the concentration range investigated. Remarkably, low concentrations of spermine and spermidine very efficiently suppress EcoRI activity. An inhibition of the EcoRI-catalyzed cleavage of DNA is also observed with NS1 and NS2, an effect that can be mimicked with other basic proteins that interact with DNA. The results are discussed in terms of the mechanism of restriction in vivo.

Bacteriophage lambda↗
PubMed1984

Structure-function relationship in Escherichia coli translational initiation factors. Characterization of IF-3 by high resolution 1H NMR spectroscopy.

Translational initiation factor-3 (IF-3) was characterized by 1H NMR spectroscopy as a function of pH and temperature and following chemical modifications. Spin-lattice relaxation times for individual resonances and bands were also measured. Several resonances were assigned to different amino acid residues by different criteria. Among these are the CH3-N of the N-terminal methionine which appears free, mobile, and very sensitive to the modification of several physicochemical parameters as well as the 3,5 and 2,6 protons of the three tyrosines (two of which play a role in the function of IF-3) which were found to be located in different magnetic environments. Two of these residues appear to be close to each other and in the vicinity of a slow reacting arginine within the tertiary structure of the factor. The properties and the titration behavior of the imidazole proton resonances suggest that the single His residue is partially buried in the protein structure. Characteristic of the IF-3 spectrum also is the presence of an abundant subset of Arg delta-CH2, Lys epsilon-CH2, and CH3 protons displaying clear cut upfield perturbations. These are probably due to the coming together of two or more apolar "fronts" which possibly arise from distant parts of the molecule and result in the close proximity between aromatic rings and aliphatic side chains. The IF-3 spectrum also includes several distinct methyl resonances significantly shifted upfield by aromatic ring currents. Overall, the characteristics of the spectrum, its relative insensitivity to temperature and ionic strength, and the existence of extensive cross-relaxation phenomena indicate that IF-3 has a highly folded tertiary structure with abundant hydrophobic regions. In spite of some heterogeneity in the distribution of the side chain environments, no indication was found for the existence of distinct domains or, at least, of extensive regions with higher mobility.

Bacterial Proteins↗
PubMed1984

Proteins from the prokaryotic nucleoid. High-resolution 1H NMR spectroscopic study of Escherichia coli DNA-binding proteins NS1 and NS2.

The 1H-NMR spectra of the two Escherichia coli basic, low-Mr (approximately equal to 9000) DNA-binding proteins NS1 and NS2 and of their native complex NS were studied at 400 MHz and a number of resonances and resonance peaks were assigned. As in the case of some eukaryotic histones, the presence of a large number of high-field perturbed Phe resonances, several shielded and deshielded methyl resonances and backbone NH protons quite inaccessible to the solvent clearly indicate the existence of extensive tertiary and, even more so, quaternary structures involving hydrophobic interactions. These structures are lost upon heating, but readily reform upon cooling. Spectral differences between NS1, NS2 and NS and the greater thermal stability of NS indicate that molecules of the heterologous subunits (NS1 and NS2) aggregate (dimerize) preferentially in comparison to the self-aggregation of the homologous subunits. Unlike those of the eukaryotic histones, the tertiary and quaternary structures of NS are insensitive to extensive variations of the ionic strength.

Bacterial Proteins↗
PubMed1984

In vitro template activity of 0.3 mRNA from wild type and initiation mutants of bacteriophage T7.

Bacteriophage T7 0.3 mRNA synthesised and processed in vitro has been purified starting from the DNA of T7+ as well as from that of two initiation mutants of T7 (CR17 with a U----C transition in the initiation codon and CR35b whose potential Shine and Dalgarno (S-D) interaction is interrupted by a G----A transition). These mRNAs were used as templates to direct the binding of fMet-tRNA and the synthesis of 0.3 protein in both E. coli and wheat germ cell-free systems. The initiation codon mutant displayed approximately 50% inhibition of fMet-tRNA binding and 0.3 protein synthesis in both systems. The S-D sequence mutant, on the other hand, was found to be less affected than the initiation triplet mutant (20%-40% inhibition) in both fMet-tRNA binding and template activity in the E. coli system. In the wheat germ system, which does not make use of the S-D interaction, however, this mutant displayed normal template activity suggesting that the inhibition obtained in the E. coli system, albeit slight, is due to the impairment of the S-D interaction and not to an alteration of the mRNA secondary or tertiary structure caused by the base substitution.

Cell-Free System↗