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Biomedical subjects

P O Olins

Publications and source records attributed to P O Olins.

29 records · Page 2Linked to original sources
PubMed1989

A novel sequence element derived from bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coli.

We recently reported that a ribosome binding site (RBS) derived from gene 10 of bacteriophage T7 (g10-L) causes a pronounced stimulation of expression when placed upstream of a variety of genes, and that this effect is probably due to a stimulation of translation efficiency in Escherichia coli (Olins, P. O., Devine, C. S., Rangwala, S. H., Kavka, K. S. (1988) Gene (Amst.) 73, 227-235). Here we present a model for the mechanism of action of the g10-L: the RBS contains a 9-base sequence which has the potential for forming a novel base-paired interaction with bases 458-466 of the 16 S rRNA of E. coli. Although such sequence homologies are rare in E. coli RBS regions, a number of similar sequences were found in the RBS regions of other bacteriophage structural genes. When an isolated homology sequence was placed upstream of a synthetic RBS, there was a 110-fold increase in the translation efficiency of the lacZ gene. Surprisingly, the homology sequence also stimulated translation when placed downstream of the initiator codon, indicating that this sequence is acting as a translational "enhancer."

Base Sequence↗
PubMed1989

Expression, purification, and in vivo activity of atrial natriuretic factor prohormone produced in Escherichia coli.

Atrial muscles of the heart are known to produce polypeptide hormones called atrial natriuretic factors (ANF) which have potent diuretic and hypotensive action. These hormones are synthesized as a larger protein precursor called pro atrial natriuretic factor or proANF which contains the biologically active ANF sequences at its C-terminus. Rat proANF (representing amino acids -1 to 128 of the coding sequence) was expressed in a soluble form in Escherichia coli. A simple purification procedure was developed which consists of boiling E. coli cell extracts in 1 M acetic acid and subjecting the supernatant to reversed-phase HPLC. The effect of intravenous administration of the purified recombinant proANF on mean arterial blood pressure was examined. The displacement dose-response curves obtained demonstrated that proANF exhibits similar, albeit less potent, physiological activity than ANF.

Amino Acid Sequence↗
PubMed1988

The T7 phage gene 10 leader RNA, a ribosome-binding site that dramatically enhances the expression of foreign genes in Escherichia coli.

Expression of foreign genes in Escherichia coli requires the juxtaposition of prokaryotic transcription and translation elements with a coding region for the foreign gene. Commonly, this results in only modest expression of the foreign gene product. Here we describe a novel ribosome-binding site (RBS; phage T7 'gene 10 leader') which is able to drive the translation of several foreign genes. This RBS dramatically enhanced the translation efficiency of all the genes we have tested to date, and was particularly effective for foreign genes. The enhanced expression was often more than 40-fold greater than that obtained using a 'consensus' RBS. A general plasmid vector has been constructed, incorporating the T7 gene 10 leader sequence, which allows the facile expression of important gene products. In this report we demonstrate the application of this system for the high-level expression of plant, mammalian and bacterial proteins in E. coli.

Base Sequence↗
PubMed1987

Phosphorylation of high- and low-molecular-mass atrial natriuretic peptide analogs by cyclic AMP-dependent protein kinase.

Synthetic high- and low-molecular-mass atrial peptides were phosphorylated in vitro by cyclic AMP-dependent protein kinase and [32P]ATP. From a series of atrial peptide analogs, it was deduced that the amino acid sequence, Arg101-Ser104 of atriopeptin was required for optimal phosphorylation. Phosphorylated AP(99-126) was less potent than the parent atriopeptin in vasorelaxant activity and receptor-binding properties. These results indicate that the presence of a phosphate group at the N-terminus of AP(99-126) decreases the interaction of the peptide with its receptor and, as a consequence, decreases bioactivity. These observations are in contrast to those of Rittenhouse et al. [(1986) J. Biol. Chem. 261, 7607-7610] who reported that phosphorylation of AP(101-126) enhanced the stimulation of Na/K/Cl cotransport in cultured vascular smooth muscle cells.

Animals↗
PubMed1987

Heparin interferes with the biological effectiveness of atriopeptin.

The chromatographic mobility of atriopeptin-28 or of the prohormone is markedly altered by preincubation of the peptides with heparin before separation on reverse-phase high performance liquid chromatography. Protamine prevented the heparin effect and reestablished the original migration pattern of the atrial peptides. The addition of heparin to either rat or human plasma samples did not interfere with the atriopeptin immunoreactivity. The influence of heparin on the biological activity of the atriopeptin-28 in anesthetized rats was also investigated. Infusion of heparin (30 U/min) significantly reduced the dose-dependent fall of blood pressure produced by atriopeptin-28, but did not interfere with the hypotensive effect of nitroglycerin. Similarly, infusion of heparin in volume-expanded rats markedly decreased the diuresis produced by atriopeptin-28 without altering the urine volume excreted in response to furosemide. These data suggest that the highly charged molecule heparin can modify the physical and biological properties of atriopeptins, perhaps by binding to the numerous arginine residues (i.e., 5 arginine residues in atriopeptin-28) in the atriopeptin molecules.

Animals↗
PubMed1986

Proteolytic processing of atriopeptin prohormone.

The metabolism of atriopeptin prohormone ANF1-126 was examined with the aid of two separate radioimmunoassays, one detecting the C-terminal atriopeptins and the other detecting a fragment of the prohormone N-terminus. Intact prohormone standards are recognized in both assays, whereas the C-terminal atriopeptins are only detected by the atriopeptin assay. Both atriopeptin and N-terminal fragment immunoreactivities were detected in rat plasma and were simultaneously elevated following intravenous administration of desamino-arginine-vasopressin. Atriopeptin immunoreactivity returned to basal levels within 60 min after desamino-arginine vasopressin administration, whereas the N-terminal fragment immunoreactivity remained elevated for more than 2 hr. Analysis of both acid-boiled and sodium dodecyl sulfate-boiled rat atrial extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed the presence of a single high molecular weight species which reacted to both antisera and which comigrated with atriopeptin prohormone standards. Western blots of plasma from desamino-arginine vasopressin-stimulated rats yielded both the low molecular weight C-terminal atriopeptin and a high molecular weight N-terminal fragment-reactive peak which was smaller than the prohormone standards and which did not possess atriopeptin immunoreactivity. A recombinant 128-amino acid atriopeptin prohormone construct, ANF1-126-Arg-Arg, was used as a model substrate for prohormone metabolism. ANF1-126-Arg-Arg was specifically cleaved followed incubation with thrombin to yield the 98-amino acid N-terminal fragment and the C-terminal atriopeptin, AP28-Arg-Arg. Processing of ANF1-126-Arg-Arg by reperfusion through an isolated heart or by incubation in serum yielded identical metabolites to those generated by incubation with thrombin. No significant metabolism was observed following incubation of the prohormone with rat plasma. We conclude that the rat heart contains the necessary enzyme to cleave both endogenous and exogenous prohormone to atriopeptin and that processing by blood enzymes is not required.

Amino Acid Sequence↗
PubMed1983

Overproduction of Escherichia coli NusA protein.

The nusA gene of Escherichia coli has been cloned into the plasmid vector pKC30 under the control of the inducible lambda pL promoter. When a strain carrying this plasmid is induced, NusA protein is overproduced more than 100-fold and constitutes 20-30% of the total cellular protein. The NusA protein purified from this strain appears identical to authentic NusA protein in its migration on SDS polyacrylamide gels and on isoelectric focusing gels. It is also able to function properly in in vitro termination and antitermination assays and in its ability to bind to E. coli core RNA polymerase.

Bacterial Proteins↗
PubMed1981

Translational regulation by ribosomal protein S8 in Escherichia coli: structural homology between rRNA binding site and feedback target on mRNA.

It has been previously shown that ribosomal protein synthesis in Escherichia coli is regulated at the level of translation by certain key ribosomal proteins. In the spc operon, S8 regulates the expression of L5 and some of the subsequent genes, while the first two genes (L14 and L24) are regulated independently. We therefore determined the DNA sequence at the junction of the L24 and L5 genes, which corresponds to the putative feedback target for S8. We show that there is a striking homology between the structure of the mRNA for this region and the known binding site for S8 on 16S rRNA. These results support the theory that the regulation of ribosomal protein synthesis is based on competition between rRNA and mRNA for regulatory ribosomal proteins.

Amino Acid Sequence↗
PubMed1981

Regulation of the S10 ribosomal protein operon in E. coli: nucleotide sequence at the start of the operon.

We have determined the DNA sequence of a 1250 base pair segment of the Escherichia coli chromosome that carries the promoter for the S10 ribosomal protein operon, the S10 gene and part of the L3 gene. A DNA fragment carrying the putative S10 promoter was cloned into the plasmid mini-Col E1, which contains a transcription termination signal close to the single Hind II site. Cells harboring the hybrid plasmid produced a relatively stable hybrid mRNA with the expected sequence, demonstrating that the promoter functions in vivo. Comparison of the mRNA sequence around the start of the S10 coding region, the presumed target site for L4 repressor protein, with the known binding site for L4 on 23S rRNA revealed the presence of sequence homologies. This supports the model of the translational feedback regulation of the S10 operon by L4.

Bacterial Proteins↗
PubMed1980

Nucleotide sequence of Scenedesmus obliquus cytoplasmic initiator tRNA.

The cytoplasmic initiator tRNA from the green alga Scenedesmus obliquus has been purified and its sequence shown to be p A G C U G A G-U m G m G C G C A G D G G A A G C G psi m G A psi G G G C U C A U t A A--C C C A U A G m G D m C A C A G G A U C G m A A A C C U Gm U C U C A--G C U A C C A-O H. The sequence has been deduced and confirmed using several different P-post labelling techniques. The sequence is similar to those of other eukaryotic cytoplasmic initiator tRNAs and it has the sequence G A U C in place of the usual G T psi C. Although it resembles lower eukaryotic species in having a U preceding the anticodon and a modified G in the T psi C stem, in overall homology it is closer to the higher eukaryotic than to the fungal initiator tRNAs.

Base Sequence↗