Solid-phase synthesis of 5'-phosphorylated oligodeoxynucleotides

We used morpholino groups to protect phosphate during the phosphorylation of the 5'-terminal ends of oligodeoxynucleotides, via phosphotriester and phosphoramidite intermediates. These groups could be removed selectively.     

Blockade by N-methylhydroxylamine or activation of guanylate cyclase and elevations of guanosine 3′,5′-monophosphate levels in nervous tissues

Hydroxylamine and N-methylhydroxylamine prevented the activation of soluble guanylate cyclase by the endogenous activator as well as by nitroso compounds such as N-methyl-N′-nitro-N-nitroguanidine or nitroprusside, while the other derivaties of hydroxylamine were ineffective. Hydroxylamine and N-methylhydroxylamine did not alter the basal guanylate cyclase activity of purified enzyme preparations. Kinetics analysis indicated that N-methylhydroxylamine competes with N-methyl-N′nitro-N-nitrosuguanidine for guanylate cyclase. The activation of guanylate cyclase by N-methyl-N′-nitro-N-nitrosoguanidine and its inhibition by N-methylhydroxylamine were reversible reactions. These efects of N-methyl-N′-nitro-N-nitrosoguanine and N-methylhydroxylamine were observed with guanylate cyclase from other tissues.N-Methylhydroxylamine preveneed the increase of guanosine 3′,5′-monophosphate (cyclic GMP) levels in cerebellar slices of guinea pig by N-methyl-N′-nitro-N-nitroguanidine, veratridine and adenosine, while the elevalations of adenosine 3′,5′-monophosphate by these agents were not affected. N-Methylhyroxylamine also blocked the increased of cyclic GMP levels by carbachol, prostaglandin E₁ and N-methyl-N′-nitro-N-nitrosoguanidine in neuroblastoma N1E 115 cells. Thus N-methylhydroxylamine prevents the activation of guanylate cyclase and the increased synthesis of cyclic GMP in responses to transmitters without blocking the synthesis of cyclic GMP via basal enzyme activity.     

A polymerization-based method to construct a plasmid containing clustered DNA damage and a mismatch

Exposure of biological materials to ionizing radiation often induces clustered DNA damage. The mutagenicity of clustered DNA damage can be analyzed with plasmids carrying a clustered DNA damage site, in which the strand bias of a replicating plasmid (i.e., the degree to which each of the two strands of the plasmid are used as the template for replication of the plasmid) can help to clarify how clustered DNA damage enhances the mutagenic potential of comprising lesions. Placement of a mismatch near a clustered DNA damage site can help to determine the strand bias, but present plasmid-based methods do not allow insertion of a mismatch at a given site in the plasmid. Here, we describe a polymerization-based method for constructing a plasmid containing clustered DNA lesions and a mismatch. The presence of a DNA lesion and a mismatch in the plasmid was verified by enzymatic treatment and by determining the relative abundance of the progeny plasmids derived from each of the two strands of the plasmid.