0000000000404381
AUTHOR
Valdis Berzins
Synthesis of recombinant atrial natriuretic peptide (rANP) using hybrid fusion protein-phage fr coat/ANP (CP/ANP).
Abstract Baumanis, V., I. Jansone, A. Skangals, I. Mandrika and V. Berzins. Synthesis of recombinant atrial natriuretic peptide (rANP) using hybrid fusion protein-phage fr coat/ANP (CP/ANP). Peptides 18(8) 1229–1235, 1997.—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 cre…
PCR-Based Site-Specific Mutagenesis
The alteration of gene structure through the substitution of specific nucleotides by site-specific mutagenesis is an important tool in modern recombinant DNA technology. Nucleotide changes are necessary not only for the analysis of the structural basis of gene and corresponding protein function, but also for the generation of novel gene products. The availability of the polymerase chain reaction (PCR) in the last decade has enabled the modification of DNA for different needs to be made more rapidly and easily than was previously possible. In the course of mutagenesis the relevant sequence changes can be introduced more readily by chemically synthesized oligonucleotide primers than by manipu…
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…
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 an…