Cloning, expression, and nucleotide sequence of a mutant glgC gene from Escherichia coli B.

A mutant glgC gene contained in a 10.9-kb PstI fragment was cloned from the Escherichia coli B strain SG5 via colony hybridization by using a wild-type glgC probe. The altered allosteric properties of the expressed ADPglucose synthetase were found to result from the conversion of proline to serine at amino acid residue 295.     

Biochemical,genetic and molecular characterization of new respiratory-deficient mutants inChlamydomonas reinhardtii

Eight respiratory-deficient mutants ofChlamydomonas reinhardtii have been isolated after mutagenic treatment with acriflavine or ethidium bromide. They are characterized by their inability to grow or their very reduced growth under heterotrophic conditions. One mutation (Class III) is of nuclear origin whereas the seven remaining mutants (Classes I and II) display a predominantly paternalmt ^- inheritance, typical of mutations residing in the mitochondrial DNA. Biochemical analysis has shown that all mutants are deficient in the cyanide-sensitive cytochrome pathway of the respiration whereas the alternative pathway is still functional. Measurements of complexes II + III (antimycin-sensitive succinate-cytochromec oxido-reductase) and complex IV (cytochromec oxidase) activities allowed to conclude that six mutations have to be localized in the mitochondrial apocytochromeb (COB) gene, one in the mitochondrial cytochrome oxidase subunit I (COI) gene and one in a nuclear gene encoding a component of the cytochrome oxidase complex. By using specific probes, we have moreover demonstrated that five mutants (Class II mutants) contain mitochondrial DNA molecules deleted in the terminal end containing the COB gene and the telomeric region; they also possess dimeric molecules resulting from end-to-end junctions of deleted monomers. The two other mitochondrial mutants (Class I) have no detectable gross alteration. Class I and Class II mutants can also be distinguished by the pattern of transmission of the mutation in crosses.Anin vivo staining test has been developed to identify rapidly the mutants impaired in cyanide-sensitive respiration.     

Crystallographic structure of metal-free concanavalin A at 2.5 Å resolution

The three-dimensional structure of demetallized concanavalin A has been determined at 2.5 Å resolution and refined to a crystallographic R-factor of 18%. The lectin activity of concanavalin A requires the binding of both a transition metal ion, generally Mn²⁺, and a Ca²⁺ ion in two neighboring sites in close proximity to the carbohydrate binding site. Large structural differences between the native and the metal-free lectin are observed in the metal-binding region and consequently for the residues involved in the specific binding of saccharides. The demetallization invokes a series of conformational changes in the protein backbone, apparently initiated mainly by the loss of the calcium ion. Most of the Mn²⁺ ligands retain their position, but the Ca²⁺ binding site is destroyed. The Ala207-Asp208 peptide bond, in the β-strand neighboring the metal-binding sites, undergoes a cis to trans isomerization. The cis conformation for this bond is a highly conserved feature among the leguminous lectins and is critically maintained by the Ca²⁺ ion in metal-bound concanavalin A. A further and major change adjacent to the isomerized bond is an expansion of the loop containing the monosaccharide ligand residues Leu99 and Tyr100. The dispersion of the ligand residues for the monosaccharide binding site (Asn14, Agr228, Asp208, Leu99, and Tyr100) in metalfree concanavalin A abolishes the lectin's ability to bind saccharides. Since the quaternary structure of legume lectins is essential to their biological role, the tetramer formation was analyzed. In the crystal (pH 5), the metal-free concanavalin A dimers associate into a tetramer that is similar to the native one, but with a drastically reduced number of inter-dimer interactions. This explains the tetramer dissociation into dimers below pH values of 6.5. © 1995 Wiley-Liss, Inc.     

Ribosomal Ribonucleic Acid-Adenine (N6-) Methylase of Escherichia coli Strain B: Ionic and Substrate Site Requirements

These investigations are concerned with the ionic and substrate-site requirements of ribosomal ribonucleic acid (rRNA)-adenine (N(6)-) methylase of Escherichia coli B. The methylase was essentially inactive in solutions of low ionic strength. The addition of MgCl(2) (optimal at 5 mM) or; to a lesser degree, KCl (optimal at 45 mM) stimulated the rate of methylation; the combination of MgCl(2) and KCl stimulated methylation to an extent equivalent to the sum of the stimulation of each acting alone. The extent of nonspecific binding of the methylase to rRNA decreased as the ionic strength of the solution increased. In the absence of ions, dimethylsulfoxide (DMSO), a nucleic acid denaturing agent, had little influence on the rate of methylation; however, DMSO plus KCl synergistically increased both the rate and the extent of methylation to a greater degree than the combination of Mg(2+) plus K(+). NH(4) (+) was less effective than K(+), and the divalent Mg(2+) offered little stimulation. Monovalent anions (acetate, nitrate, and chloride) were equally effective, whereas divalent SO(4) (2-) was decidedly inhibitory. The appropriate ionic milieu of mono- and divalent cations was required to provide the appropriate conformation of the rRNA and to facilitate specific interactions of the methylase and its recognition sites in the rRNA, while decreasing nonspecific ionic binding of the methylase to rRNA. DMSO may facilitate methylation by increasing the number of substrate sites exposed in single-stranded regions of the rRNA. Nonmethylatable rRNA species served as competitive inhibitors, whereas the polyanions deoxyribonucleic acid, transfer RNA, and polyadenylic acid were inactive. Micrococcus lysodeikticus and Bacillus subtilis rRNA, methylated by the methylase, each contained two distinct heptanucleotides containing newly synthesized 6-methyladenine moieties. The data are consistent with the view that E. coli strain B possesses two species of rRNA-adenine (N(6)-) methylases, each of which recognizes a specific adenine moiety in a unique pentapurine nucleotide sequence in a single-stranded region of rRNA.     

Characterization of S-Adenosylmethionine: Ribosomal Ribonucleic Acid-Adenine (N6-) Methyltransferase of Escherichia coli Strain B

This study is concerned with the isolation and characterization of the enzyme, S-adenosylmethionine:ribosomal ribonucleic acid-adenine (N⁶⁻) methyl-transferase [rRNA-adenine (N⁶-) methylase] of Escherichia coli strain B, which is responsible for the formation of N⁶-methyladenine moieties in ribosomal ribonucleic acids (rRNA). A 1,500-fold purified preparation of the species-specific methyltransferase methylates a limited number of adenine moieties in heterologous rRNA (Micrococcus lysodeikticus and Bacillus subtilis) and methyl-deficient homologous rRNA. The site recognition mechanism does not require intact 16 or 23S rRNA. The enzyme does not utilize transfer ribonucleic acid as a methyl acceptor nor does it synthesize 2-methyladenine or N⁶-dimethyladenine moieties. Mg²⁺, spermine, K⁺, and Na⁺ increase the reaction rate but not the extent of methylation; elevated concentrations of the cations inhibit markedly. The purified preparations utilize 9-β-ribosyl-2,6-diaminopurine (DAPR) as a methyl acceptor with the synthesis of 9-β-ribosyl-6-amino-2-methylaminopurine. A comparison of the two activities demonstrated that one methyltransferase is responsible for the methylation of both DAPR and rRNA. This property provides a sensitive assay procedure unaffected by ribonucleases and independent of any specificity exhibited by rRNA methyl acceptors.     

Studies on haemoglobin immobilized by cross-linking with glutaraldehyde. Cross-linked soluble polymers and artificial membranes

Human haemoglobin was immobilized by cross-linking with glutaraldehyde as soluble polymers and artificial membranes. Effects of pH and 2,3-diphosphoglycerate on oxygen binding and cross-linking were studied with haemoglobin immobilized in both the oxy and deoxy states. The cooperativity is suppressed and the affinity is increased when compared with native haemoglobin. Haemoglobin immobilized in the oxy state exhibited a higher oxygen affinity than that immobilized in the deoxy state. The alkaline Bohr effect is not significantly different from that of native haemoglobin. The 2,3-diphosphoglycerate influence on oxygen binding was reduced by one third with immobilization. In order to separate the chemical and the "conformation freezing' effects on the properties of immobilized haemoglobin, glutaraldehyde-modified haemoglobin in oxy and deoxy states was produced. Oxygen binding was studied and chemical modifications were checked by electrophoresis and gel filtration. This chemically modified haemoglobin without polymerization and without intra-chain bridging exhibits a behaviour similar to that of cross-linked soluble polymers or membranes of haemoglobin.     

The role of intracellular lumina in the repolarization process of a colonic adenocarcinoma cell line

When glucose is added to the culture medium, some cells of the undifferentiated HT-29 line derived from a human colonic adenocarcinoma develop spherical structures, demonstrated to be intracellular by the ruthenium red staining method, which are bordered with microvilli, contain osmiophilic substances and resemble intracellular lumina. When glucose is replaced by galactose in the culture medium, the cells differentiate apical membranes bordered with microvilli. Our observations suggest that these new apical membranes correspond to the membranes of intracellular lumina which have opened outside the cells. We suggest that intracellular lumina may represent "compensation" for loss of polarity of epithelial cells and may be an important step in the repolarizing process of the cells.     

Significance of dinucleoside tetraphosphate production by cultured tumor cells exposed to the presence of ethanol

The use of 30 to 50% ethanol solutions to extract the nucleotides from HTC and A-459 cells results in dinucleoside tetraphosphate (Ap4X) levels 3-30-fold as high as those obtained by 5% classical trichloracetic acid extraction, while ATP levels are identical in both cases. The amplification factor varies with the percentage of ethanol and duration of contact between the cells and the extraction mixture. It remains constant for the HTC cells during cell growth, but exhibits a maximum for the A-459 cells towards the end of the exponential growth period. The incorporation of radioactivity in Ap4X when [alpha-32P]ATP is added to the extraction mixture suggests an Ap4X neosynthesis in the presence of ethanol. The results carried out in the presence of pyrophosphate, EDTA and zinc acetate strongly suggest that aminoacyl-tRNA synthetases could be responsible for the increase in Ap4A content with ethanol treatment. Nevertheless, the effect of ethanol is probably not the result of an activation of these enzymes, but rather, as already suggested by earlier results in our laboratory, the result of a fast inactivation of the degradation enzymes.