Isolation, characterization and molecular cloning of Duplex-Specific Nuclease from the hepatopancreas of the Kamchatka crab

<p>Abstract</p> <p>Background</p> <p>Nucleases, which are key components of biologically diverse processes such as DNA replication, repair and recombination, antiviral defense, apoptosis and digestion, have revolutionized the field of molecular biology. Indeed many standard molecular strategies, including molecular cloning, studies of DNA-protein interactions, and analysis of nucleic acid structures, would be virtually impossible without these versatile enzymes. The discovery of nucleases with unique properties has often served as the basis for the development of modern molecular biology methods. Thus, the search for novel nucleases with potentially exploitable functions remains an important scientific undertaking.</p> <p>Results</p> <p>Using degenerative primers and the rapid amplification of cDNA ends (RACE) procedure, we cloned the Duplex-Specific Nuclease (DSN) gene from the hepatopancreas of the Kamchatka crab and determined its full primary structure. We also developed an effective method for purifying functional DSN from the crab hepatopancreas. The isolated enzyme was highly thermostable, exhibited a broad pH optimum (5.5 – 7.5) and required divalent cations for activity, with manganese and cobalt being especially effective. The enzyme was highly specific, cleaving double-stranded DNA or DNA in DNA-RNA hybrids, but not single-stranded DNA or single- or double-stranded RNA. Moreover, only DNA duplexes containing at least 9 base pairs were effectively cleaved by DSN; shorter DNA duplexes were left intact.</p> <p>Conclusion</p> <p>We describe a new DSN from Kamchatka crab hepatopancreas, determining its primary structure and developing a preparative method for its purification. We found that DSN had unique substrate specificity, cleaving only DNA duplexes longer than 8 base pairs, or DNA in DNA-RNA hybrids. Interestingly, the DSN primary structure is homologous to well-known Serratia-like non-specific nucleases structures, but the properties of DSN are distinct. The unique substrate specificity of DSN should prove valuable in certain molecular biology applications.</p

Effects of Carrageenans on Biological Properties of Echinochrome

Sea urchin pigment echinochrome A (Ech), a water-insoluble compound, is the active substance in the cardioprotective and antioxidant drug Histochrome&#174; (PIBOC FEB RAS, Moscow, Russia). It has been established that Ech dissolves in aqueous solutions of carrageenans (CRGs). Herein, we describe the effects of different types of CRGs on some properties of Ech. Our results showed that CRGs significantly decreased the spermotoxicity of Ech, against the sea urchin S. intermedius sperm. Ech, as well as its complex with CRG, did not affect the division and development of early embryos of the sea urchin. Ech reduced reactive oxygen species production (ROS) in neutrophils, caused by CRG. The obtained complexes of these substances with pro- and anti-activating ROS formation properties illustrate the possibility of modulating the ROS induction, using these compounds. The CRGs stimulate the induction of anti-inflammatory IL-10 synthesis, whereas Ech inhibits this synthesis and increases the production of the pro-inflammatory cytokines IL-6 and TNF&#945;. The inclusion of Ech, in the complex with the CRGs, decreases Ech&#8217;s ability to induce the expression of pro-inflammatory cytokines, especially TNF&#945;, and increases the induction of anti-inflammatory cytokine IL-10. Thus, CRGs modify the action of Ech, by decreasing its pro-inflammatory effect. Whereas, the Ech&#8217;s protective action towards human epithelial HT-29 cells remains to be unaltered in the complex, with &#954;/&#946;-CRG, under stress conditions