Research activity

Before 1990 the research activity of the Department of Gene Regulation (formerly Department of Genetic Engineering) was focused on gene synthesis, optimization of heterologous gene expression, development of technologies for production of recombinant human hormones and cytokines, investigation of structure and heterogeneity of human and mammalian repetitive DNAs.

After 1990 the Dept. Gene Regulations runs project in the following research areas: 1) Initiation and enhancement of translation in prokaryotes; 2) Post-translational alterations of proteins in bacteria; 3) Molecular mechanisms of action of biologically active proteins; 4) Physico-chemical studies on recombinant proteins and nuclei acids ; 5) Bioinformatics.

The investigation of initiation of translation and enhancement of translation in bacteria led to the discovery of (non-Shine-Dalgarno) initiators of translation and therefore of new (alternative) mechanism of initiation of this process.

The new initiators of translation were found among already known enhancers of translation (such as the e and the w sequences), or in the non-translated regions of some eukaryotic genes (such as the Phytolacca americana antiviral protein, PAP, gene?), or isolated from combinatorial DNA libraries. These sequences were complementary to different domains in 16S rRNA and did not share any homology with the consensus SD sequence.

Studying post-translational alterations of recombinant proteins obtained from bacteria, we found that these proteins, as well as the host bacterial proteins were subjected to non-enzymatic glycosylation (glycation). Glycation is a chemical reaction of reducing sugars and other carbonyl compounds with the primary amino groups in proteins. It starts with formation of Schiff bases, which are converted consecutively into Amadori products and advanced glycation end products (AGEs). Once initiated, this reaction proceeds spontaneously and leads to chemical proteolysis (fragmentation of the polypeptide chain), accumulation of heterocyclic compounds (AGEs) and loss of biological activity. This reaction is found to affect both recombinant and host proteins (and even nucleic acids) in bacteria and is described for the first time in prokaryotes. The research activities of the team are now directed towards identification of bacterial glycating compounds, factors affecting glycating activity in bacteria, inhibitors of glycation, glycation – genetic instability relationship, etc.

To study structure-function relationship, two recombinant proteins – human interferon gamma (hIFNg) and Phytolacca americana antiviral protein (PAP) were mutated. The antiviral activity assays showed that the C-terminal part of hIFNg consisting of 21 aminoacid residues (out of 143 AA) is dispensable for its biological activity. The stepwise deletion of the C-terminal aminoacids resulted in the beginning in the gradual increase in antiviral activity (up to 10 fold after removing the first 9 residues). Further deletions up to 21 aminoacids correlated with a decrease in antiviral activity and led to a complete lost of activity after deleting of more than 21 C-terminal aminoacids.

The mutation analysis of PAP showed that the first 90 N-terminal aminoacids (out of 209) are dispensable for its antiviral activity.

The physico-chemical studies on biopolymers (proteins and nucleic acids) includes: identification of adducts formed by non-enzymatic glycosylation; spectroscopic and thermodynamic studies on aggregation and folding-unfolding of recombinant proteins; mechanisms of non-enzymatic hydrolysis of proteins and nucleic acids; new fluorescent dyes for nucleic acid staining, etc.

The bioinformatic studies started after 2000 and are now carried out in two directions: i) analysis of genetic information available from the DNA databases and ii) modeling of biomolecular structures and biological processes.

The Escherichia coli genome was analyzed for content and distribution of codon pairs. This analysis revealed that the codon pairs are non-randomly distributed and that their frequency or occurrence varies from zero to 4600 times. Thus 19 missing codon pairs were identified, 17 of which represented combinations between sense and stop codons. Equations are derived from estimation of the modulating (enhancing or attenuating) effect of codon pairs on gene expression. Analyses are now in progress to compare codon usage on all sequenced up to now bacterial and mitochondrial genomes.

Modeling structures of important biopolymers, the first model of electrostatic interactions in the molecule of hIFNg was created in collaboration with Dr. A. Karshikoff from Karolinska Institute in Stockholm