Research

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.

Alternative translation initiation in bacteria

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.

Maillard reaction in bacteria

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.

Bioinformatics and in silico modeling

The bioinformatics 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 human interferon-gamma (hIFN gama was created in collaboration with Dr. A. Karshikoff from Karolinska Institute in Stockholm.

Development of hIFN gama based pharmaceuticals

We have developed two pharmaceutical formulations – eye drops for treatment of eyes’ viral infections (Gammaferon) and gel (Virogel) for treatment of Herpes zoster. Both medicines were registered for production and sale in Bulgaria. Another direction of department research is linked to the possibility hIFN gama derivatives to be used for treatment of autoimmune disorders. Current investigations show that autoimmune diseases such as multiple sclerosis, alopecia aerates, myasthenia gravis and autoimmune uveitis are associated with increased IFN gama levels. Post-transplant atherosclerosis is also linked to the overproduction of IFN gama. As a result the graft becomes rejected due to blockage of the blood vessels. The new approach we are developing consists of competitive inhibition of the endogenous IFNg ama activity through IFN gama analogues that can bind to the cognate receptor but are devoid of biological activity. We have created such IFN gama analogues using a site directed mutagenesis of the IFN gama gene. To date, over 100 IFN gama mutants have been tested and some of them demonstrate an encouraging results in competitive studies as far as they lower the IFN gama activity in model studies in vitro.

Molecular ecology of microbial communities

This is a new direction of the department’s investigations linked to the application of genomic tools for studying the diversity, distribution and functioning of microbial communities in extreme habitats. The investigations are focused on phylogenetics, taxonomy and shift in ecology and structure of microbial communities in environmental settings such as soil and water polluted with heavy metals, marine sediments contaminated by waste water, alpine lakes and hot thermal springs.