title: Enzymatic addition of modified cytosine nucleotides to DNA. Methacrylate polymerization by an azo pyrimidine.
abstract: An azo pyrimidine that induces the polymerization of 1-glyceryl methacrylate has been prepared. 1-Glyceryl methacrylate contains substituent glycol groups for binding heavy atoms for subsequent electron microscopic studies. Oxodation of 1-methyl N4-ureidocytosine (compound 1A) with N-bromosuccinimide produced 4-aminocarbonylazo-2-pyrimidinone (compound 2A). This orange compound shows a broad peak (lambda max = 349 nm) in its ultraviolet absorption spectrum. Mixing compound 2A with mildly acidic aqueous solutions of 1-glyceryl methacrylate (pH 3.6) resulted in polymerization of the methacrylate. Under these conditions, the ester 1-glyceryl methacrylate does not hydrolyze appreciably as judged by oxidation of the glycol groups with NaIO4 and spectrophotometric analysis of the HCHO liberated using Schiff's reagent. Attachment of azo nucleotides to the 3'-terminus of DNA was achieved in two steps. First, N4-ureidocytosine deoxynucleotides were enzymatically added to single strand DNA primers using calf thymus terminal deoxynucleotidyl transferase, Co2+, and N4-ureido-2'-deoxycytidine 5'-triphosphate (prepared by the HSO3--catalyzed transamination reaction of dCTP with semicarbazide). Second, these modified nucleotides were oxidized with N-bromosuccinimide to produce DNA that contained azo pyrimidine nucleotides at its 3'-end (azoDNA). Upon adding acid to the azoDNA, the azo nucleotides decomposed. If these nucleotides induce methacrylate polymerization upon decomposition as did compound 2A, it may be possible to mark the location of DNA termini in situ for electron microscopy by attaching heavy atoms to the poly(1-glyceryl methacrylate) formed. Such studies may elucidate the nature and location of the ends of the eukaryotic chromosomal DNA molecule in both chromosomes and interphase nuclei.
mesh_terms: D000180:Acrylic Resins; D055598:Chemical Phenomena; D002621:Chemistry; D003596:Cytosine / Q000031:analogs & derivatives*; D004247:DNA; D004253:DNA Nucleotidylexotransferase; D005989:Glycerides; D011109:Polymethacrylic Acids; D011744:Pyrimidinones; D013056:Spectrophotometry, Ultraviolet
chemical_list: C026516:4-aminocarbonylazo-2-pyrimidinone; D000180:Acrylic Resins; D005989:Glycerides; D011109:Polymethacrylic Acids; D011744:Pyrimidinones; C026517:1-methyl-N(4)-ureidocytosine; C009932:polyglycerylmethacrylate; D003596:Cytosine; D004247:DNA; D004253:DNA Nucleotidylexotransferase