By Bernard N. Fields and Rudolf Jaenisch (Eds.)
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LAFEMINA AND GARY S. HAYWARD molecules labeled S1 and S4) and those that do not fork (S3, S8 and S10). All of these longer duplex structures were probably formed by interstrand annealing between two complementary fragments. The two molecules consisting of an S-loop and short duplex stem, the spur, an adjacent long duplex stem Figure 1 : Tracings from electron micrographs of "stemloop" structures formed by self-annealing single strands of human CMV(Towne) DNA. The DNA at low concentration was denatured with alkali, neutralized and incubated at 68° for 2 hr with slow cooling.
To determine whether these clones contained identical viral sequences, the recombinant plasmids were digested with Hind III. 7 X 10 6 daltons) from the Eco RI site. The number of Hind III sites A. 1 2 3 X B, 1 2 3 X FIGURE 4. Analysis of cloned fragments representing HCMV band 13 with A. Eco RI and B. Hind III restriction endonucleases. The resulting fragments were separated on agarose gels and stained with ethidium bromide. Lane 1, clone 2; 2, clone 19; and 3, clone 20. 2 X 1 0 6 ) . 3. MOLECULAR CLONING OF CYTOMEGALOVIRUS GENOME 31 within the viral insert can be determined from the number of fragments produced upon restriction endonuclease digestion of the recombinant plasmid.
Panel B shows clones co-migrating with HCMV bands 8b, 8c, 9, 10, 11a and lib. Bands lld-g, 12 and 13 are represented by the clones shown in panel C and the smallest cloned inserts, corresponding to bands 14a, 14b, 15, 16, 17, and 18, appear in panel D. Figure 3E shows an example of the hybridization of 32 P-labeled HCMV DNA to a Southern blot of the DNA from panel D. The sensitivity of the hybridization method under these conditions is demonstrated by the appearance of a band in lane 5 of panel E, even though there is no trace of DNA by ethidium bromide staining (lane 5 of panel D ) .