Addition of poly(adenylic acid) to RNA using polynucleotide phosphorylase: an improved method for electron microscopic visualization of RNA-DNA hybrids.
We have observed that the enzyme polynucleotide phosphorylas from M. luteus or from E. coli will polymerize adenosine (A) from adenosine diphosphate onto 3' ends of RNA molecules. For gene mapping, the poly(A)-tailed RNA is hybridized to its complementary sequence on a longer DNA strand. The position of the poly(A)tail, and thus the position of the 3' end of the RNA on the DNA strand, can then be observed by electron microscopy. Our preferred mapping technique involves the synthesis of a poly(A)-specific label by polymerization of a poly(dBrU) tail onto one or both ends of a linear duplex DNA of defined length (a restriction fragment) and hybridization of this label to the poly(A) tail. In test experiments with a plasmid containing a Drosophila DNA sequence coding for 5S rRNA genes, overall labeling efficiencies of 70--80% were achieved.
D000818:Animals; D004262:DNA Restriction Enzymes; D004331:Drosophila melanogaster; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008837:Micrococcus / Q000201:enzymology; D008854:Microscopy, Electron; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011061:Poly A; D011117:Polyribonucleotide Nucleotidyltransferase / Q000378:metabolism*; D012335:RNA, Ribosomal
D012335:RNA, Ribosomal; D011061:Poly A; D011117:Polyribonucleotide Nucleotidyltransferase; D004262:DNA Restriction Enzymes