1991 - BS/MS, Kiev State University, Kiev, Ukraine
1996 - Ph.D., Institute of Molecular Biology and Genetics, Kiev, Ukraine
1997 - 2003 - Postdoctoral training, University of Texas at Austin and European Molecular Biology Laboratory, Heidelberg, Germany

There are two main directions of our current research: advanced genome engineering using tailor-made site-specific DNA recombinases and cell replacement in tissues using genetically modified adult stem cells. We are also interested in applying site-specific DNA recombination systems to synthetic biology.


Position for a postdoc is available
Positions for Ph.D. students are available

Advanced genome engineering

The goal of this research direction is developing new tools to manipulate genomes. We develop these tools using variants of site-specific recombinases evolved to recognize chosen sites in a genome of interest. These pre-existed genomic sites resemble to some extent the native recombination sequences for wild-type recombinases. To evolve the variants we use target-linked molecular evolution approaches: site-directed and random mutagenesis and DNA shuffling. In our research we mainly use yeast site-specific recombinases: Flp, TD1, R1 and others.

Examples of genomic sequences that resemble native recombination target for Flp recombinase, FRT (A): FRT-like sequences in human interleukin-10 gene (B) and LTRs of HIV-1 (C). These FRT-like sequences were identified using developed computer program TargetFinder in the corresponding genomic regions. The positions within the FRT-like-‘binding elements’ that match those in FRT (A) are denoted by upper case green letters. Red lower case letters indicate mismatch. TargetFinder scores for the FRT-like sites, normalized to a score of 100 for FRT, are shown at the right.

FRT-like sequences that have relativey high resemblance to FRT can be found in a genome quite often: every about 10 kb.

Model gene targeting in E. coli using the evovled Flp variant FV7. This variant was evolved to recombine FRT-like sequences in human IL10 gene. (A) schematics of the assay, (B) image of a plate after the targeting, (C) control digestion of the plasmid DNA isolated from white (w) or blue (b) colonies.

Testing FV7 in mammalian cells (CHO). Deletion of EGFP gene flanked by FRT and FRT-like DNA sequence from human IL10 gene (FL-IL10A) by Flp variant FV7 leads to expression of DsRed gene (reaction is diagrammed at the top). Tests were performed in CHO cells transiently transfected with the reporter plasmid (A) and in CHO cells, which have the reporter integrated into genome (B). 48 hours after transfection residual fluorescence of EGFP is seen in the cells, in which recombination took place; this reflects either incomplete recombination (in A) or long half-life of EGFP in CHO cells (A, B). After cells were split and colonies formed, no EGFP fluorescence was detected in the cells expressing DsRed.

Cell replacement

New direction of our research is replacement of old/senescent/dysfunctional cells in human tissues using adult stem cells. We are interested in developing approaches to engineer adult stem cells to express certain genes with therapeutic properties, to replace certain genes with their allelic variants known to extend the life span or to prevent early onset of aging disorders, and to repair genetic defects in the stem cells by replacing defective genomic DNA with the ‘wild type’ one. These genetically engineered stem cells could then be used to replace cells in adult tissues. Currently we are working on replacing ‘old’ albino melanocytes in the hair bulbs of albino mice with the ‘new’ ones, in which ability to produce melanin is restored.

A C57BL/6J mouse treated with antibodies ACK45 to deplete amplifying populations of melanoblasts after hair depilation that initiated hair cycle. Since melanocytes in the hair bulbs in the treated areas are depleted, the hair that grows after depilation is white.

Genetic networks

Site-specific recombination systems can be applied to design programmable genetic networks. In such networks site-specific recombinases can be used to change connectivity between the network components or to create bits of information. Genes for site-specific DNA recombinases and functional/regulatory DNA fragments flanked by corresponding recombination sites can be incorporated into genetic networks as detachable modules to create multifunctional networks with predictable behavior. These networks can be used in the emerging field of synthetic biology to design new pathways and developing new ways to perform logical or computational operations.

Site-specific recombination systems can be used to design programmable genetic networks. (A) Site-specific recombinase can invert a DNA segment flanked by the recombination sites in the head-to-head orientation. (B) Genetic networks can utilize different arrangements of genes, regulatory elements and recombination sites. (C) Computational genetic network (simplified). The corresponding elements of the network are placed on separate DNA molecules to allow compact design, ease of handling, and replacement and expansion of the elements. A controlling module regulates transcription of the genes of the computational module, which, in turn, regulate expression of the reporter module. P, promoter; O, operator; SSR, gene for site-specific DNA recombinase; lacZ, reporter gene.

Selected publications

• Bolusani, S., Ma, C., Paek, A., Konieczka, J. H., Jayaram, M., Voziyanov, Y.
  Evolution of variants of yeast site-specific recombinase Flp that utilize native genomic sequences as recombination target sites.
  Nucleic Acids Res. (2006) 34, 5259-5269.
  
  
• Konieczka J.H., Paek A., Jayaram M., Voziyanov Y.
  Recombination of hybrid target sites by binary combinations of Flp variants: mutations that foster inter-protomer collaboration and enlarge substrate tolerance
  J. Mol. Biol. (2004) 339, 365-378
  
  
• Voziyanov Y., Konieczka J.H., Stewart A. F., Jayaram M.
  Stepwise manipulation of DNA specificity in Flp recombinase: Progressively adapting Flp to individual and combinatorial mutations in its target site
  J. Mol. Biol. (2003) 326, 65-76
  
  
• Voziyanov Y., Stewart A.F., Jayaram M.
  A dual reporter screening system identifies amino acid at position 82 in Flp site-specific recombinase as a determinant for target specificity
  Nucleic Acids Res. (2002) 30, 1656-63
  
  
• Voziyanov Y., Pathania S., Jayaram M.
  A general model for site-specific recombination by the integrase family recombinases
  Nucleic Acids Res. (1999) 27, 930-941
  
  
• Lee J., Voziyanov Y., Pathania S., Jayaram M.
  Structural alterations and conformational dynamics in Holliday junctions induced by binding of a site-specific recombinase
  Mol. Cell (1998) 1, 483-493
  
  
• Voziyanov Y., Lee J., Whang I., Lee J., Jayaram M.
  Analyses of the chemical step in Flp site-specific recombination: Synapsis may not be a pre-requisite for strand cleavage
  J. Mol. Biol. (1996) 256, 720-735