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J Alves

Publications and source records attributed to J Alves.

At least 55 records · Page 3Linked to original sources
PubMed1990

Probing the function of individual amino acid residues in the DNA binding site of the EcoRI restriction endonuclease by analysing the toxicity of genetically engineered mutants.

We have developed an assay that allows analysis of the activity of EcoRI restriction endonuclease (ENase) and its mutants in vivo. This assay is based on the fact that wild type (wt) EcoRI ENase is toxic for Escherichia coli cells not expressing the EcoRI methyltransferase (MTase). The viability factor defined by the ratio of the viable counts of E. coli cultures having or not having expressed the ecoRIR gene for a defined time is 10(-6) for wt EcoRI ENase and close to one for a totally inactive EcoRI ENase mutant. While the EcoRI MTase (M.EcoRI) provides substantial protection against the toxic effects of the wt EcoRI ENase and several of the mutants, some mutants become more toxic in the presence of M.EcoRI. Twenty-four different DNA-binding-site mutants of EcoRI ENase were characterized in their activity in vivo with this assay. The results obtained allow us to conclude that the structural integrity of the region at and around aa 200 seems to be very critical for the enzymatic function of EcoRI ENase: nonconservative replacements there lead to viability factors of 1-10(-2). While our results indicate that the region around aa 144 and 145 is also involved in the EcoRI ENase-catalyzed reaction, it is also evident that the effects of mutation there are not as large: viability factors of approx. 10(-3) are obtained even for drastic replacements. These results are discussed in the light of the x-ray structure analysis of an EcoRI ENase-DNA recognition complex.

Amino Acid Sequence↗
PubMed1989

Fluorescence stopped-flow kinetics of the cleavage of synthetic oligodeoxynucleotides by the EcoRI restriction endonuclease.

We have investigated in fluorescence stopped-flow and temperature-jump experiments the EcoRI-catalyzed cleavage of synthetic palindromic tridecadeoxynucleotides which contain the EcoRI site but differ in the flanking sequences. The overall reaction can be resolved in several reactions which were analyzed by a nonlinear least-squares fitting procedure on the experimental data. The result of this analysis is a minimal scheme that describes the overall reaction in terms of the rate constants of the individual reactions. According to this scheme EcoRI and the tridecadeoxynucleotide substrates associate in the presence of Mg2+ in a nearly diffusion-controlled process. This is followed by a reaction which is or includes the cleavage of the first phosphodiester bond. There is no indication for a time-resolved conformational transition prior to catalysis. After cleavage of the first strand, dissociation of the nicked double strand can occur, which then rearranges to the original palindromic double-stranded substrate and is bound again by the enzyme. Alternatively, the nicked double strand can be cleaved in the second strand. This reaction is followed by product release from the enzyme. The magnitude of the individual rate constants depends on the substrate used; the differences explain the preference of EcoRI for substrates that contain AT as compared to GC base pairs next to the recognition site.

Base Sequence↗
PubMed1989

Genetic engineering of EcoRI mutants with altered amino acid residues in the DNA binding site: physicochemical investigations give evidence for an altered monomer/dimer equilibrium for the Gln144Lys145 and Gln144Lys145Lys200 mutants.

We have genetically engineered the Arg200----Lys mutant, the Glu144Arg145----GlnLys double mutant, and the Glu144Arg145Arg200----GlnLysLys triple mutant of the EcoRI endonuclease in extension of previously published work on site-directed mutagenesis of the EcoRI endonuclease in which Glu144 had been exchanged for Gln and Arg145 for Lys [Wolfes et al. (1986) Nucleic Acids Res. 14, 9063]. All these mutants carry modifications in the DNA binding site. Mutant EcoRI proteins were purified to homogeneity and characterized by physicochemical techniques. All mutants have a very similar secondary structure composition. However, whereas the Lys200 mutant is not impaired in its capacity to form a dimer, the Gln144Lys145 and Gln144Lys145Lys200 mutants have a very much decreased propensity to form a dimer or tetramer depending on concentration as shown by gel filtration and analytical ultracentrifugation. This finding may explain the results of isoelectric focusing experiments which show that these two mutants have a considerably more basic pI than expected for a protein in which an acidic amino acid was replaced by a neutral one. Furthermore, while wild-type EcoRI and the Lys200 mutant are denatured in an irreversible manner upon heating to 60 degrees C, the thermal denaturation process as shown by circular dichroism spectroscopy is fully reversible with the Gln144Lys145 double mutant and the Gln144Lys145Lys200 triple mutant. All EcoRI endonuclease mutants described here have a residual enzymatic activity with wild-type specificity, since Escherichia coli cells overexpressing the mutant proteins can only survive in the presence of EcoRI methylase. The detailed analysis of the enzymatic activity and specificity of the purified mutant proteins is the subject of the accompanying paper [Alves et al. (1989) Biochemistry (following paper in this issue)].

Base Sequence↗
PubMed1989

Changing the hydrogen-bonding potential in the DNA binding site of EcoRI by site-directed mutagenesis drastically reduces the enzymatic activity, not, however, the preference of this restriction endonuclease for cleavage within the site-GAATTC-.

According to the X-ray structure analysis of an EcoRI-oligodeoxynucleotide complex [McClarin et al. (1986) Science 234, 1526], sequence specificity is mediated by 12 hydrogen bonds, 6 from each of the two identical subunits of the dimeric enzyme to the recognition site -GAATTC-: Arg200 forms two hydrogen bonds with guanine, while Glu144 and Arg145 form four hydrogen bonds to adjacent adenine residues. Changing the hydrogen-bonding potential at the recognition site without perturbing the rest of the interface should lead to the recognition of degenerate sequences [Rosenberg et al. (1987) in Protein Engineering (Oxender, D. L., & Fox, C. F., Eds.) pp 237-250, Liss, New York]. We have shown previously that replacing Glu144 by Gln and Arg145 by Lys affects the activity of the enzyme, not, however, its specificity [Wolfes et al. (1986) Nucleic Acids Res. 14, 9063]. We show now that also the mutation of Arg200 to Lys, the double mutation Glu144Arg145 to GlnLys, and the triple mutation Glu144Arg145Arg200 to GlnLysLys do not lead to a detectable degeneracy of the specificity of cleavage by EcoRI but significantly impair the catalytic activity of this enzyme. A detailed analysis of the steady-state kinetics of cleavage of pUC8 DNA and a tridecadeoxynucleotide substrate demonstrates that the reduction in activity for all DNA binding site mutants investigated so far is mainly due to a decrease in kcat, with the exception of the Arg200 to Lys mutant, which is only impaired in its KM.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1988

Spectroscopic and hydrodynamic studies reveal structural differences in normal and transforming H-ras gene products.

We have recorded the circular dichroism spectra of the cellular and the viral H-ras gene products both in the absence and in the presence of guanine nucleotides and analyzed these spectra in terms of the secondary structure composition of these proteins. It is shown that the GTP complex of the ras proteins has a different secondary structure composition than the GDP complex and, furthermore, that there are differences in the secondary structure of the viral ras protein and the cellular ras protein. We have also recorded and analyzed the circular dichroism spectrum of the isolated guanine nucleotide binding domain of the Escherichia coli elongation factor Tu (EF-Tu), which has been considered as a model for the tertiary structure of the ras proteins [McCormick, F., Clark, B. F. C., LaCour, T. F. M., Kjeldgaard, M., Norskov-Lauritsen, L., & Nyborg, J. (1985) Science (Washington, D.C.) 230, 78-82]. Our data show that the guanine nucleotide binding domain of EF-Tu (30% alpha-helix and 16% beta-pleated sheet for the GDP complex) has quite a different secondary structure composition than the ras proteins (e.g., the cellular ras protein has 47% alpha-helix and 22% beta-pleated sheet for the GDP complex), indicating that the protein core comprising the guanine nucleotide binding site might be similar but that major structural differences must exist at the portion outside this core. Normal and transforming ras proteins also differ slightly in their hydrodynamic properties as shown by sedimentation velocity runs in the analytical ultracentrifuge.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Transformation, Neoplastic↗
PubMed1986

Site directed mutagenesis experiments suggest that Glu 111, Glu 144 and Arg 145 are essential for endonucleolytic activity of EcoRI.

We have constructed a plasmid (pRIF 309+) carrying the EcoRI restriction endonuclease gene and the f1 origin of replication. Upon transformation of this plasmid into E. coli and infection with bacteriophage f1 single stranded plasmids are produced which can be used for sequencing and site directed mutagenesis. Using this single stranded DNA and synthetic oligodeoxynucleotides we have introduced point mutations at defined positions of the EcoRI gene. Since in pRIF309+ the EcoRI gene is under the control of the pL-promoter, high level expression of the mutated EcoRI gene could be obtained upon induction. Mutant EcoRI enzymes were purified to homogeneity and characterized in structural and functional terms. Our results demonstrate that the Glu 111----Gln, Glu 144----Gln and Arg 145----Lys -mutants adopt a very similar conformation as the wild type enzyme, but have by two orders of magnitude smaller specific activities than the wild type enzyme, mainly due to a reduction of the Vmax-value.

Base Sequence↗
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

The influence of sequences adjacent to the recognition site on the cleavage of oligodeoxynucleotides by the EcoRI endonuclease.

We have investigated the influence of the nucleotide sequence adjacent to the recognition site on the rate of cleavage of DNA by the restriction endonuclease EcoRI. For this purpose two decadeoxynucleotides, d(G-G-G-A-A-T-T-C-T-T) (Ia) and d(A-A-G-A-A-T-T-C-C-C) (Ib) were synthesized. The duplex Ia X Ib is cleaved by EcoRI preferentially in the dA-rich strand (approximately 10 times over the dG-rich strand). The individual nucleotides Ia and Ib are also cleaved by EcoRI, Ib at a higher rate than Ia and both at a lower rate than Ia X Ib. The temperature dependence of the reaction rate shows that only double-stranded oligodeoxynucleotides are substrates for the EcoRI endonuclease. We have, furthermore, synthesized oligomers of d(G-G-A-A-T-T-C-C), which contain two, three and four EcoRI sites, respectively. These oligodeoxynucleotides are preferentially cleaved at the sites next to the 5' end, where the recognition site is only flanked by one dG X dC base pair, in contrast to the other sites which are flanked by three such pairs. These data indicate that sequences adjacent to the recognition site influence the rate of cleavage: dA X dT base pairs enhance and dG X dC base pairs slow down the hydrolytic activity of the EcoRI endonuclease.

Base Composition↗
PubMed1983

Does the specific recognition of DNA by the restriction endonuclease EcoRI involve a linear diffusion step? Investigation of the processivity of the EcoRI endonuclease.

The time course of the EcoRI endonuclease catalysed cleavage of three substrates, two plasmid DNAs and one oligonucleotide, each with two EcoRI sites, was measured. The two plasmid DNAs with the EcoRI sites 318 and 96 base pairs apart are cut in a distributive fashion, while the oligonucleotide with the EcoRI sites 8 base pairs apart is cut in a partially processive manner. It is concluded that a linear diffusion of the EcoRI endonuclease on its substrate across long stretches of DNA is not likely to be operative during the recognition process. Microscopic dissociation-reassociation processes, however, increase the probability of the enzyme to attack further sites located in the immediate vicinity of a given site.

Base Composition↗
PubMed1982

Two identical subunits of the EcoRI restriction endonuclease Co-operate in the binding and cleavage of the palindromic substrate.

The cleavage of radioactively labelled double-stranded d(G-G-A-A-T-T-C-C) was studied in single turnover experiments with substrate and enzyme both being in the micromolar range. The reaction rate was found to increase with enzyme concentration until a ratio of one tetrameric enzyme to two double-stranded substrates was reached, further increase of the enzyme concentration then leads to a sharp decline of the reaction rate. These findings are interpreted in the following manner. (a) Two subunits of the EcoRI endonuclease co-operate in binding and possibly also in cleaving the palindromic substrate. (b) The enzymatic action of the EcoRI endonuclease is inhibited by excess enzyme, possibly due to unspecific binding of the enzyme-substrate complex. The self-inhibition of EcoRI endonuclease has also been observed with macromolecular substrates.

Binding, Competitive↗
PubMed1975

[Emergencies in internal medicine. General management in coma situations].

One of the most important aspects of the Emergency Medicine is the coma status as much as intra and extrahospitalary ambient. The interest of om study is based according the hardness of anamnesis, and the severity of symptoms induced by a altered conscience and by the need of a emergency therapy. This work summarized the general semiology of the patient in order to diagnostic, severity and etiology of the coma. The general behaviour therapeutic and some criterions about an irreversibility is also studied.

Coma↗
PubMedDate not supplied

[Upper gastrointestinal endoscopy in children: indications and results]

OBJECTIVE: To present and discuss the indications, the endoscopic and histologic findings and the complications of upper digestive endoscopies carried out in children and adolescents.METHODS: We have done 228 endoscopies in pediatric patients with ages between 3 months to 19 years (x=5.7 y) during a period of 8 months. General anesthesia was used in 80% of patients (182). Biopsies were done in 210 patients and the Helicobacter pylori was searched for in 130 patients by antral biopsies with Hematoxylin-Eosin coloration.RESULTS: The endoscopy indication was diagnostic in all patients, and in 18 cases there was some therapeutic procedure. Fifty-eight percent of examinations presented endoscopic alteration and 84% had abnormal histologic findings. The most frequent endoscopic findings were esophagitis in 50 patients, gastritis in 44 and duodenitis in 29. Ten peptic ulcers were diagnosed. Between the histologic findings the most frequent ones were gastritis in 87 patients, esophagitis in 50 and atrophy of duodenal mucosa in 9. The H. pylori was positive in 25 (19.2%) patients.CONCLUSION: The upper digestive endoscopy has become an essential procedure to the pediatric gastroenterology practice, amplifying the available diagnostic means and enabling therapeutic endoscopy in the presence of upper digestive lesions. In the present report, 58% of the studied population presented some endoscopic lesion.

Journal Article↗