| Abstract [eng] |
Transposons have been widely used in experiments of genome analyzing and directional rearrangements in vivo, and lately in vitro, since they had been discovered. In this work Tn-Km transposon was modified providing unique targets of restriction endonucleases MreI, Bsp1407I, BseXI, AarI within inverted repeats. The efficiency of transposition of modified transposons was established. The results have shown that transposons with identical inverted repeats (MreI, Bsp1407I or BseXI) are as effective as their progenitor Tn-Km. However, mosaic transposons having different inverted repeats are less effective in transposition reaction. The usefulness of modified transposon Tn-Km-Mre was evaluated by performing random mutagenesis of eco47IR gene. The other task of this work was to find out those regions of the encoded Eco47I restriction endonuclease which tolerate small insertions. Four such regions were identified. Based on this information five positive selection cloning vectors of new generation were constructed: pJET1-3A, pJET1-1MCS, pJET1-2MCS, pJET1-3MCS, pJET1-4MCS. In addition, plasmid pJET1-3A was tested for its cloning properties and found to work very well. It was also shown that 12 amino acid residues located at the very N terminus of Eco47I are not essential for functional activity of this restriction endonuclease. Therefore pJET1-1MCS can’t be used as a universal positive selection cloning vector, otherwise inserted DNA fragments that will accidentally provide codon for the initiator methionine would not be cloned. |
