Biomedical subjects

V Berzins

Publications and source records attributed to V Berzins.

6 recordsLinked to original sources

PubMed1998

Long-range translational coupling in single-stranded RNA bacteriophages: an evolutionary analysis.

In coliphage MS2 RNA a long-distance interaction (LDI) between an internal segment of the upstream coat gene and the start region of the replicase gene prevents initiation of replicase synthesis in the absence of coat gene translation. Elongating ribosomes break up the repressor LDI and thus activate the hidden initiation site. Expression studies on partial MS2 cDNA clones identified base pairing between 1427-1433 and 1738-1744, the so-called Min Jou (MJ) interaction, as the molecular basis for the long-range coupling mechanism. Here, we examine the biological significance of this interaction for the control of replicase gene translation. The LDI was disrupted by mutations in the 3'-side and the evolutionary adaptation was monitored upon phage passaging. Two categories of pseudorevertants emerged. The first type had restored the MJ interaction but not necessarily the native sequence. The pseudorevertants of the second type acquired a compensatory substitution some 80 nt downstream of the MJ interaction that stabilizes an adjacent LDI. In one examined case we confirmed that the second site mutations had restored coat-replicase translational coupling. Our results show the importance of translational control for fitness of the phage. They also reveal that the structure that buries the replicase start extends to structure elements bordering the MJ interaction.

Base Sequence↗

PubMed1998

The natural 6 S RNA found in Q beta-infected cells is derived from host and phage RNA.

The RNA of Escherichia coli infected with RNA bacteriophage Q beta was isolated and screened for replicable short-chained RNA. In contrast to earlier assumptions we show that (i) short-chained replicable RNA is a very minor part of the RNA synthesized in the infection cycle, and (ii) that the replicable RNA isolated from infected cells is derived from cellular RNA, in particular 23 S rRNA and 10 Sa RNA, and from Q beta RNA itself. None of the many RNA species known from in vitro experiments was found. The RNA species isolated were all inefficient templates. No replicable RNA could be isolated from non-infected cells. Even in cells expressing high amounts of Q beta replicase very few RNA species could be isolated. RNA generated in vitro in template-free synthesis is therefore not derived from RNA species found in vivo, and replicable RNA found in vitro is generated by a mechanism fundamentally different from the one operating in vivo.

Allolevivirus↗

PubMed1998

A long-range interaction in Qbeta RNA that bridges the thousand nucleotides between the M-site and the 3' end is required for replication.

The genome of the positive strand RNA bacteriophage Qbeta folds into a number of structural domains, defined by long-distance interactions. The RNA within each domain is ordered in arrays of three- and four-way junctions that confer rigidity to the chain. One such domain, RD2, is about 1,000-nt long and covers most of the replicase gene. Its downstream border is the 3' untranslated region, whereas upstream the major binding site for Qbeta replicase, the M-site, is located. Replication of Qbeta RNA has always been puzzling because the binding site for the enzyme lies some 1,500-nt away from the 3' terminus. We present evidence that the long-range interaction defining RD2 exists and positions the 3' terminus in the vicinity of the replicase binding site. The model is based on several observations. First, mutations destabilizing the long-range interaction are virtually lethal to the phage, whereas base pair substitutions have little effect. Secondly, in vitro analysis shows that destabilizing the long-range pairing abolishes replication of the plus strand. Thirdly, passaging of nearly inactive mutant phages results in the selection of second-site suppressor mutations that restore both long-range base pairing and replication. The data are interpreted to mean that the 3D organization of this part of Qbeta RNA is essential to its replication. We propose that, when replicase is bound to the internal recognition site, the 3' terminus of the template is juxtaposed to the enzyme's active site.

Allolevivirus↗

PubMed1997

Rapid evolution of translational control mechanisms in RNA genomes.

We have introduced 13 base substitutions into the coat protein gene of RNA bacteriophage MS2. The mutations, which are clustered ahead of the overlapping lysis cistron, do not change the amino acid sequence of the coat protein, but they disrupt a local hairpin, which is needed to control translation of the lysis gene. The mutations decreased the phage titer by four orders of magnitude but, upon passaging, the virus accumulated suppressor mutations that raised the fitness to almost wild-type level. Analysis of the pseudorevertants showed that the disruption of the local hairpin, controlling expression of the lysis gene, had apparently been so complete that its restoration by chance mutations could not be achieved. Instead, alternative foldings initiated by the starting mutations were further stabilized and optimized. Strikingly, in the pseudorevertants analyzed, translational control of the lysis gene had been restored. This feat was accomplished by, on average, four suppressor mutations that generally occurred at codon wobble positions. We also introduced 11 mutations in a hairpin more upstream in the coat protein gene and not implicated in lysis control. Here the titer dropped by three logs, but pseudorevertants with a fitness close to wild-type were soon generated. These pseudorevertants again were the result of the optimization of alternative foldings induced by the mutations. The transition of the secondary structure from wild-type to pseudorevertant could be visualized by structure probing. Our study shows that the folding of the RNA is an important phenotypic property of RNA viruses. However, its distortion can easily be overcome by optimizing alternative base-pairings. These new structures are not qualitatively equivalent to the original one, since they do not successfully compete with the wild-type.

Base Sequence↗

PubMed1997

Synthesis of recombinant atrial natriuretic peptide (rANP) using hybrid fusion protein-phage fr coat/ANP (CP/ANP).

Recombinant atrial natriuretic peptide (rANP) was expressed in and isolated from E. coli. rANP was purified using HPLC. Amino acid analysis, partial sequencing, and molecular mass were determined. Fused protein was used to rise polyclonal antibodies and to develop of immunoenzymatic assays of rANP and CP/ANP. Experiments were designed to study rANP effects on isolated rabbit aortic strips and to examine hypotensive, diuretic, and natriuretic activity, as well as renal creatinine clearance, in an in vivo rat model. Identity of recombinant and commercial ANP has been confirmed. Physiological activity of CP/ANP has allowed the investigators to predict the conformation of CP/ANP, pro-ANP processing, and the method by which fusion protein interacts with ANP receptors.

Animals↗

PubMed1993

High level expression of alpha-human atrial natriuretic factor as a fusion polypeptide with phage fr coat protein in Escherichia coli.

A synthetic DNA sequence coding for the 28 amino acid residues of alpha-human atrial natriuretic factor (alpha-hANF) and the N-terminal linker tripeptide Ile-Asp-Lys was inserted in the 3'-terminal part of the RNA bacteriophage fr coat protein gene. The cloned hybrid gene was isolated and placed into an expression vector under the control of the inducible E. coli tryptophan promoter and phage fr coat protein translation initiation region (TIR) sequence. In an appropriate host strain the expressed fusion protein accounts for at least 10% of the total cellular protein. In order to achieve high-cell density in a bioreactor while maintaining efficiency of alpha-hANF expression, improved cultivation conditions were selected using modified Shielach-Bauer's culture media containing glucose, yeast extract and bacto tryptone at an initial concentration of 2 g l-1 of each, adding concentrate of medium throughout the microbial growth and maintaining the dissolved oxygen in a range of 25-30%. At 13-14 h cultivation, the cell density reached 40 g cell dry weight per liter and the yield of fusion protein exceeded 45 mg g-1 cell dry weight. Fusion protein from solubilized E. coli cells was purified to homogeneity by ion exchange chromatography on DEAE-, CM-cellulose, QAE Sephadex A25 columns and selective precipitation.

Amino Acid Sequence↗