POLY(A).RNA GENE MAPPING IN EM VOL. 17, NO. 18, 1978 3883 Addition of Poly( adenylic acid) to RNA Using Polynucleotide Phosphorylase: An Improved Method for Electron Microscopic Visualization of RNA-DNA Hybrids? James Douglas Engel and Norman Davidson* ABSTRACT: We have observed that the enzyme polynucleotide phosphorylase from zyxwvutsrqpo 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 ob- served by electron microscopy. Our preferred mapping tech- E l e c t r o n microscopic mapping of the position of RNA- DNA hybrid regions is a valuable method for determining the positions of genes on defined DNAs. One method in use in this laboratory involves the attachment of an electron opaque label such as ferritin (Angerer et al., 1976; Hershey et al., 1977; Yen et al., 1977; Broker et al., 1978) or polymer spheres (Manning et al., 1975; Sodja and Davidson, 1978) to the 3’ end of the RNA to assist in visualization of the RNA-DNA hybrid. This general approach, while effective, has some disadvantages. Several complex synthetic steps are necessary for the prepa- ration of the modified RNA and the derivatized electron opaque label. More importantly, the overall efficiency of la- beling is usually less than 50%. An efficient simple method of mapping poly(A) segments on RNA molecules has been developed (Wensink et al., 1974; Hsu et al., 1973; Bender and Davidson, 1976). The technique used in this laboratory involves polymerization of poly(dT) tails onto internal 3’ termini of nicked circular duplex DNAs, such as SV40 DNA, with the enzyme terminal deoxynucleotidyl- transferase. The poly(dT) pairs with the poly(A) on the RNA; the circular duplex DNA is a readily recognized label in the electron microscope. It was later shown that poly(dBrU) is a still better affinity reagent for reaction with poly(A) tails, because the stability of A-BrU base pairs is greater than that of A-T base pairs (W. W. Bender, personal communication). The A-T and A-BrU association reactions are rapid and re- quire only a slight excess of poly(dT) or poly(dBrU) to poly(A). Used in this way, a labeling efficiency of greater than 75% is achieved for poly(A) ends on natural poly(A)-con- taining RNAs (Bender and Davidson, 1976). In order to extend this simple and efficient gene-mapping approach, we have developed techniques for adding poly(A) tails to the 3’-OH end of RNAs-for example, tRNA, 5s RNA or poly(A)- mRNA-that do not naturally have a poly(A) tail. We find that the enzyme polynucleotide phos- phorylase will add zyxwvutsrqp A residues (from adenosine diphosphate) onto the 3’ ends of all RNA molecules tested. Furthermore, From the Department of Chemistry, California Institute of Tech- nology, Pasadena, California 91 125. zyxwvutsrqpo Received March zyxwvutsrq 4, 1978. Contri- bution number 5759. This work was supported by a Helen Hay Whitney Postdoctoral Fellowship (to J.D.E.) and by Grant GM10991 from the United States Public Health Service (to N.D.). 0006-2960/78/04 17-3883$01 .OO/O nique 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 ex- periments with a plasmid containing a Drosophila DNA se- quence coding for 5s rRNA genes, overall labeling efficiencies of 70-80% were achieved. our tests indicate a high efficiency of gene mapping with these RNA molecules. We also report on the preparation of linear restriction fragments with one or two poly(dBrU) tails at the ends. These labels are particularly useful for the kinds of mapping exper- iments described here. Materials and Methods Chemicals. rADP, r[3H]ADP, and oligo(dT)-cellulose (T3) were purchased from Boehringer-Mannheim, Schwarz-Mann, and Collaborative Research, respectively. Enzymes. Polynucleotide phosphorylase (PNPaseI zy ) prep- arations were the generous gifts of Drs. T. Colburn and J. Dodgson (E. coli B) and C. Klee (M. luteus). The E. coli PNPase (from J.D.) had been purified through the Sephadex G-200 step (Dodgson and Wells, 1977); the polymerization activity was 600 pmol of rADP incorporated per 15 min per A280 protein. The M. luteus PNPase-I (primer independent) was purified as described (Klee and Singer, 1968) and had an activity of approximately 300 pmol of rADP incorporated (at 37 “C) per 15 min per A280 protein; the PNPase-T (primer dependent) was obtained by mild trypsin treatment of PNPase-I and then blocking sulfhydryl groups with N-ethyl- maleimide (Klee, 1968); PNPase-T activity was approximately 200 pmol of rADP incorporated per 15 min per A280 enzyme. Terminal deoxynucleotidyltransferase (“minimal nuclease”) was purchased from P-L Biochemicals. Restriction endonuc- leases EcoR1, HindIII, and BamH1 were purchased from New England Biolabs. Bacterial alkaline phosphatase (BAPF) was purchased from Worthington Biochemicals. Gel Electrophoresis. Neutral agarose (Helling et al., 1974), 8% acrylamide, 98% formamide (Maniatis et al., 1975), and CH3HgOH-agarose slab gels (Bailey and Davidson, 1976) were run as described in the figure legends and text. Abbreviations used are: nt, nucleotides (chain length); PNPase, polynucleotide phosphorylase from either E. coli or M. luteus (EC 2.7.7.8) (specified in text); divalent and monovalent labels, poly(BrU) polymerized onto one or both ends of a plasmid DNA restriction fragment (see text); kb, kilobase (1000 nucleotides); Tris, zyxw 2-amino-2-hydroxymethyl-1,3- orooanediol; EDTA, (ethylenedinitri1o)tetraacetic acid; DEAE, dieth- ylaminoethyl; Pipes, 1,4-piperazinediethanesulfonic acid; Tes, 2-[[2- hydroxy-I ,I-bis(hydroxymethyl)ethyl]amino]ethanesulfonic acid. 0 1978 American Chemical Society