Specific DNA Restriction Fragments as Internal Markers in the Electron Microscope: Superimposing the ɸX174 Genetic Map on ɸX174/S13 DNA Heteroduplexes

Citation

Compton, John Lee (1975) Specific DNA Restriction Fragments as Internal Markers in the Electron Microscope: Superimposing the ɸX174 Genetic Map on ɸX174/S13 DNA Heteroduplexes. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/nr2r-8c58. https://resolver.caltech.edu/CaltechTHESIS:09152021-165317789

Abstract

Chapter I

ɸX174 RF II* molecules were constructed in vitro by annealing circular ɸX174 viral DNA and linear ɸX174 complementary strand DNA. When a specific fragment of ɸX174 RF purified from a digestion with the restriction endonuclease Hind from Hemophilus influenzae was included in the renaturation mixture ɸX174 RF II molecules containing a loop corresponding in size to the fragment were seen. This was confirmed for three different DNA fragments.

ɸX174 RF II molecules were constructed in the presence of two restriction fragments. In addition to molecules containing one loop corresponding in size to one of the fragments molecules containing two loops were seen. The two loops corresponded in size to the two restriction fragments and the distances between the two loops corresponded to the distances between the two fragments on the Hind cleavage map of ɸX174 RF.

By including two fragments of distinguishable size that map asymmetrically on the cleavage map in the renaturation mixture, ɸX174 RF II molecules were constructed which could be oriented on the cleavage map. Using the two loops as internal markers the ɸX174 genetic map was superimposed on the electron micrographs of these molecules.

Chapter II

ɸX174/S13 DNA heteroduplexes were constructed by annealing purified ɸX174 complementary strand DHA 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 ɸX174 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 ɸX174 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 ɸX174 cleavage map.

The ɸX174 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 double-stranded: two regions of about 10% of the ɸX174 genome each in genes A and H and a region of about 25% of the ɸX174 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.

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