Isolation, cloning and molecular characterization of genes encoding single-strand specific endonucleases from Parsley (Petroselinum crispum L.)

Abstract

Single-strand specific (sss) nucleases (S1/P1 family) are widely used in molecular genetics studies. The most widely used sss-nucleases are those plant endonucleases with the ability of specific cleavage of mismatch loops in heteroduplex DNA molecules. Because of the valuable activity of these endonucleases, enzymatic cleavage of heteroduplex DNA molecules is one of the most common techniques of point mutation detection in genomes. In this study, gene sequences encoding single-strand specific endonucleases were isolated from parsley (Petroselinum crispum L.) using heterologous primers in PCR reactions. The isolated gene sequences, after cloning and molecular identification, were designated as pars I and pars II. Physicochemical, structural and phylogenetic analysis using software tools, verified PARS I and PARS II as two members of S1/P1 family with a substantial homology to heteroduplex DNA specific endonucleases. However, it is essential to produce pure enzyme preparations as native or recombinant forms to determine mechanisms of activity and substrate preferences of these nucleases on heteroduplex DNA substrates.

Keywords

• single-strand specific nucleases
• petroselinum crispum
• enzymatic cleavage of heteroduplex dna
• cloning