The effects of DNA intercalators on chromatin of chicken red blood cells——differential extraction on nonhistone proteins

Taking advantage of the effects on DNA secondary structure of two DNA-intercalators,ethidium bromide and chloroquine,we used each of them to treat nuclei from both mature erythrocytes and reticulocytes of chicken,as an alternative approach to study the relationships between DNA secondary structure,nuclear proteins and chromatin structure.We presented results of differential extraction of nuclear proteins from nuclei with DNA-intercalators,as well as preliminary characterization of these proteins.A 45kd protein is the major component in fractions extracted by both intercalators from nuclei from either mature erythrocytes or reticulocytes and seems to be a DNA-binding protein.Furthermore,from current concepts of functional aspects of DNA conformation and structural heterogeneity in chromatin and nuclear proteins,we have discussed both the significance of our results as well as technical aspects of this approach.     

The araBAD operon of Salmonella typhimurium LT2. II. Nucleotide sequence of araA and primary structure of its product, L-arabinose isomerase

The nucleotide sequence of gene araA of Salmonella typhimurium LT2 has been determined. The gene encodes an L-arabinose isomerase (EC 5.3.1.4) of 500 amino acid residues with a calculated Mr of 55814. The ATG start codon of araA is 10 bp distal to the TAA termination codon of araB. A presumed ribosome-binding site (RBS) "TAAGGA" 7 bp from the ATG codon overlaps the stop codon of araB. L-Arabinose isomerase was purified and the amino acid composition is in agreement with that predicted from the DNA sequence. The NH2-terminus of the protein is modified as the sequence cannot be analyzed by the automated Edman degradation. Amino acid composition analyses of both NH2-terminal and C-terminal cyanogen bromide (CNBr) cleaved peptides and partial amino acid sequence of the C-terminal peptide are consistent with the deduced amino acid sequence.     

Stable gene amplification in the chromosome of Bacillus subtilis

We constructed five different structures, consisting of a genetic marker flanked by directly repeated sequences 2-4 kb long, in the Bacillus subtilis chromosome. When a selective pressure was applied amplification of the marker and one of the repeats was observed in all cases. Amplification was not detected with two markers which were not flanked by the repeated sequences. The maximum amplification level observed with the different structures varied between 5 and 50. The size of the most amplified structure corresponded to 7.5% of the chromosome. Amplification was stable upon growth of cells under non-selective conditions. Each copy of an amplified gene was expressed with equal efficiency. These results indicate that chromosomal gene amplification may be useful for constructing genetically engineered B. subtilis strains.     

Isolation of a human tissue-type plasminogen-activator genomic DNA clone and its expression in mouse L cells

We have isolated a cDNA clone corresponding to a substantial portion of the human tissue-type plasminogen activator (t-PA) protein. It encodes almost all of the protein B chain and part of the 3' untranslated region. We have used this clone to screen bacteriophage lambda and cosmid libraries of human genomic DNA. Several related genomic clones were isolated. One of these, a cosmid clone, carried approx. 40 kb of human DNA. Mapping experiments indicate that the region containing the protein-coding exons is approx. 20 kb in length. The cosmid, containing the t-PA gene and the aminoglycosyl-3'-phosphotransferase dominant-selection marker, was introduced into mouse L cells. Approximately half of the transformants were shown to produce human t-PA. We demonstrated that the fibrinolytic t-PA activity could be specifically quenched by anti-t-PA antibody and that the recombinant t-PA was of similar size (by SDS-polyacrylamide gel electrophoresis) to the t-PA produced by the human Bowes melanoma cell line. Our results suggest that the cosmid clone carries the whole t-PA coding region together with the regulatory elements necessary for its expression.     

Molecular cloning using immune sera of a 22-kDal minor outer membrane protein of Vibrio cholerae

Using antisera prepared against live Vibrio cholerae we have selected several recombinant DNA clones, plasmids pPM440, pPM450 and pPM460, encoding the gene for a 22-kDal V. cholerae peptidoglycan-associated-outer-membrane protein. This is a minor protein in V. cholerae but is expressed in large amounts when the cloned gene is present in Escherichia coli K-12, where it is exposed on the cell surface as judged by ELISA. We have localized the gene within the cloned DNA by transposon mutagenesis and deletion analysis followed by analysis of whole cells and minicells to identify the plasmid-encoded proteins. The DNA region encoding the protein seems to be conserved between El Tor and Classical strains as judged by Southern DNA hybridization.     

Interaction of membrane surface charges with the reconstituted ADP/ATP-carrier from mitochondria

Various modulating influences of negative and positive membrane charges on binding and transport properties of the reconstituted ADP/ATP carrier from mitochondria were investigated. The results are interpreted in terms of functional and structural asymmetries of the adenine nucleotide carrier embedded in the liposomal membrane. The surface potential of liposomes was measured directly either by potential-dependent adsorption of the fluorescent dye $\text{2-p-}\text{toluidinylnaphthalene}$ 6-sulfonate (TNS) or by the $\text{p}\text{K}$ shift of the lipophilic pH indicator pentadecylumbelliferone. These results were correlated with the following observations. (1) Negative surface potentials increase the apparent dissociation constant, $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$, for binding of the negatively charged inhbitor carboxyatractylate to the reconstituted carrier protein. (2) Surface potentials modulate the apparent transport affinity, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, of the reconstituted adenine nucleotide carrier for ADP and ATP. The interaction of surface charges with the transport function was investigated with carrier proteins oriented both right-side-out and inside-out. Thus the influence of the surface potential on the function of the ADP/ATP carrier could be determined for the internal and external active sites of the translocator on the outer side of the membrane. Large discrepancies were observed not only between the potentials measured directly (fluorescent dyes) and those measured indirectly (binding and transport affinities), but also between the different surface potentials determined from the influence on the alternatively oriented carrier proteins. The effect of surface charges was rather weak on the cytosolic side of the translocator, whereas there was a strong influence of surface charges on the active site at the matrix side. The most obvious explanation, i.e., screening of negative membrane charges by positively charged amino acid residues at the protein surface, could be ruled out. Besides the modulation of binding affinities for substrates and inhibitors, an additional side-specific effect of surface charges on the transport velocity was observed. Again, the influence on the internal active site of the ADP/ATP carrier was found to be much higher than that on the cytosolic site. The observed effects can be explained by a definite structural asymmetry of the carrier embedded in the liposomal membrane. That site which is physiologically exposed to the cytosol is located at a considerable distance from the plane of the membrane, whereas the opposite site seems to be in close proximity to the membrane surface. Moreover, a spatial equivalence of carboxyatractylate binding site and nucleotide binding site at the external side of the carrier protein was concluded.     

Localization of the two free thiol groups in the porcine pancreatic alpha-amylase I sequence

Porcine pancreatic alpha-amylase I, a single 496 residue long polypeptide chain, contains 5 disulfide bridges and 2 free -SH groups. The conditions for specific blocking of native amylase either with radioactive N-ethyl maleimide or with labeled iodoacetic acid were determined. Under these conditions 2 moles of blocking reagent are incorporated per mole of amylase. [14C]-S-succinimido amylase was cleaved by CNBr and the resulting peptides were purified. Only one of them the CNBr 2 + 3 peptide (178 residues) was found labeled. Ts1 a 33-residue peptide containing the whole radioactivity was purified from the tryptic digest of this large fragment. After reduction and carboxymethylation Ts1A, (22 residues) was obtained which contains 2 moles of succinyl-Cys and one mole of CM-Cys per mole of peptide. Chymotryptic digestion of Ts1A yielded 2 equally labeled peptides: C1 (16 residues) and C2 (6 residues). Automated sequencing of both peptides and counting of the PTH-amino acids shows that the free cysteines are only 15 residues apart in the sequence.     

A biochemical study of the reconstitution of d-lactate dehydrogenase-deficient membrane vesicles using fluorine-labeled components

Fluorine-19 labeled compounds have been incorporated into lipids and proteins of Escherichia coli. ¹⁹F-Labeled membrane vesicles, prepared by growing a fatty acid auxotroph of a d-lactate dehydrogenase-deficient strain on 8,8-difluoromyristic acid, can be reconstituted for oxidase and transport activities by binding exogenous d-lactate dehydrogenase. ¹⁹F-Labeled d-lactate dehydrogenases prepared by addition of fluorotryptophans to a tryptophan-requiring strain are able to reconstitute d-lactate dehydrogenase-deficient membrane vesicles. Thus, lipid and protein can be labeled independently and used to investigate protein-lipid interactions in membranes.