Aligning the φX174 genetic map and the φX174/S13 heteroduplex denaturation map

Abstract

φ X174/S13 DNA heteroduplexes were constructed by annealing purified φ X174 complementary strand DNA and S13 viral DNA. The heteroduplexes were mounted for electron microscopy under a series of increasingly denaturing conditions and photographed. The micrographs were measured and oriented to form a map showing the progressive denaturation of various regions of the molecule. Such heteroduplex molecules were totally denatured and mixed with a denatured specific fragment of φ X174 RF ‡ purified from a digestion with the restriction endonuclease Hind of Hemophilus influenzae . After renaturation the molecules were mounted for electron microscopy under standard conditions. Each of three different φ X174 RF fragments could be seen in a different, specific region of the heteroduplex and could be oriented with respect to the others to correspond to the known φ X174 cleavage map. ‡ Abbreviations used: RF, double-stranded circular replicative form DNA; RF I, supercoiled RF DNA with both strands covalently closed: RF II, RF, DNA with one or more single-stranded nicks in either strand. The φ X174 genetic map was thus superimposed on the heteroduplex denaturation map by using the included fragments as reference points. Under mildly denaturing conditions three regions of the heteroduplex are conserved as doublestranded: two regions of about 10% of the φ X174 genome each in genes A and H and a region of about 25% of the φ X174 genome covering gene E and most of gene F . Under increasingly denaturing conditions only two small duplex regions of 2 and 4% are conserved, one each in genes E and F .