The use of primed synthesis by DNA polymerase I to study an intercistronic sequence of phiX-174 DNA.
A decadeoxynucleotide complementary to ten nucleotides in the major ribosome-protected fragment of phiX-174 plus-strand DNA has been chemically synthesized and used as a primer for DNA polymerase I on phiX-174 plus-strand DNA as template. The sequence of the first 40 nucleotides incorporated onto the decadeoxynucleotide has been determined. This sequence extends further the sequence of the intercistronic region preceding gene G and shows the presence of another termination codon. The sequence was determined by using manganese as the activating cation for DNA polymerase I which allows ribonucleotides to be incorporated as well as deoxyribonucleotides. The ribo-substituted product was then cleaved specifically at the ribonucleotide residues to generate a series of overlapping ribo-terminated fragments whose sequences were sufficient to determine the complete sequence of the first 40 nucleotides. No evidence for misincorporation by DNA polymerase I in the presence of manganese was detected.
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D003090:Coliphages / Q000378:metabolism*; D003597:Cytosine Nucleotides / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004279:DNA, Viral / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology; D006160:Guanosine Triphosphate / Q000378:metabolism; D008345:Manganese / Q000494:pharmacology; D009841:Oligonucleotides / Q000138:chemical synthesis; D013698:Templates, Genetic; D013942:Thymine Nucleotides / Q000378:metabolism
D003597:Cytosine Nucleotides; D004279:DNA, Viral; D009841:Oligonucleotides; D013942:Thymine Nucleotides; D008345:Manganese; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D004254:DNA Nucleotidyltransferases