DNA methylation: sequences flanking C-G pairs modulate the specificity of the human DNA methylase.

Synthetic single-stranded oligodeoxynucleotides of known sequence have been used as in vitro substrates for a partially purified HeLa cell DNA methylase. Although most oligonucleotides tested cannot be used by the HeLa DNA methylase in vitro, we have found a unique 27mer, containing 2 C-G pairs, that is an excellent substrate for the enzyme. Analysis of the methylation of the 27mer, its derivatives and other oligomer substrates reveal that the HeLa DNA methylase does not significantly methylate an oligomer which contains just one C-G pair. In addition, only one of the two C-G pairs in the 27mer is methylated and this methylation is abolished if the other C-G pair is converted to a C-A pair. Furthermore, the HeLa enzyme apparently cannot methylate C-G pairs located in compounds containing a high A + T content. The most efficient methylation occurs with multiple separated C-G pairs in a compound with a high G + C content (greater than 65%). The results suggest that clustering of C-G pairs in regions of the DNA high in G + C content may be the preferred site for DNA methylation in vivo.

Synthesis and pharmacological evaluation of a series of analogues of 1-methylisoguanosine.

A series of analogues of the pharmacologically active marine natural product 1-methylisoguanosine (1) was evaluated for biological activity in muscle relaxant, cardiovascular, antiinflammatory, and antiallergic tests. Modifications at the 1 position produced the ethyl, n-butyl, n-octyl, and phenyl derivatives 3-6, respectively. Substitutions at the 8 position provided the bromo, hydrazino and amino compounds 9-11. Modification at the 5' position yielded the deoxy, iodo, and phosphate derivatives 15, 13, and 16, as well as the cyclic 3',5'-phosphate 17. The synthesis of the C-nucleoside analogue 19 was achieved from the beta-D-ribofuranosylcarboximidic ester 20. The acyclic analogue 29 and the beta-D-arabinofuranosyl derivative 35 were both synthesized by reaction of methyl isocyanate with the appropriately protected aminocyanoimidazole precursors 28 and 32. 1-Methylxanthosine (12), isoguanosine (7), and 2-methoxyadenosine (18) were also synthesized. At doses up to 100 mg/kg po, the 5'-phosphate 16, cyclic 3',5'-phosphate 17, and the O-methylated analogue 2-methoxyadenosine 18 were active in producing muscle relaxation and hypothermia. These compounds possessed antiallergic activity and produced dose-dependent falls in mean blood pressure and heart rate as did the 1-ethyl (3) and 1-n-butyl (4) analogues. In general, antiinflammatory activity paralleled the other results, except that the cyclic 3',5'-phosphate 17 was inactive at the dose tested, while the 3,5'-anhydronucleoside 14 was weakly active and displayed antiallergic effects.

Synthesis of arabinofuranosyl derivatives of 3-deazaguanine.

Synthesis of four arabinofuranosyl derivatives of the antitumor agent 3-deazaguanine is described. By the use of 13C and 1H nuclear magnetic resonance spectroscopy, the structures of these nucleosides were established to be alpha and beta pairs of N-7 and N-9 arabinosides of 3-deazaguanine. In contrast to 3-deazaguanine and its ribosyl derivative, the nucleosides described in this paper were found to be inactive against Sarcoma 180 in mice at 100 mg/kg.

Chemical synthesis of two deoxyribododecanucleotides for the attachment of restriction termini to an artificial minigene.

In order to permit in vivo cloning of an artificial minigene designed to code for a modified S-peptide, the phosphodiester method for the chemical synthesis of two dodecadeoxyribonucleotides is described. Each of the latter possesses antiparallel complementarity to one of the two minigene strands and to the single-stranded EcoRI-generated end. They can thus serve as cohesive termini ("splints") for polynucleotide ligase joining.