Volume 3

Gene expression / Benjamin Lewin.

  • Lewin, Benjamin.
Date:
1974-1977
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Gene expression / Benjamin Lewin. Source: Wellcome Collection.

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    spread for electron microscopy. They are first mixed with a basic protein, usually cytochrome c, which binds to the nucleic acid and gives a struc ture that can conveniently be visualized (for review see Davis, Simon and Davidson, 1971). The nucleic acid-protein preparation may be spread in either of two ways. In aqueous spreading medium, duplex nucleic acid sequences appear as elongated threads but single strands suffer random interactions and thus become condensed into bushes. This identifies the positions of the single stranded regions relative to the duplex regions, whose contour lengths can be measured. In spreading medium containing appropriate concentrations of formamide and salt, the random collapse of the single strands is prevented and so both duplex and single strand regions form elongated threads. The single strand regions appear thinner in diameter than the duplex regions, and so both single strand and duplex contour lengths can be measured. Heteroduplex mapping is a particularly appropriate technique to use for defining sequences of the sex factor because so many F' variants are available to provide suitable preparations of parental molecules that are in part identical and in part different. In the experiments of Sharp et al. (1972), covalently closed circles of F and F' molecules were nicked to introduce single breaks in each strand, the duplexes were denatured in NaOH and single strands recovered, and then the F preparation was annealed with the F' preparation. Heteroduplexes form whenever an F strand renatures with a complementary F' strand and were visualized by spreading in a medium containing 50 /xg/ml cytochrome c and 45% form- amide. Two types of heteroduplex structure can be predicted, depending on whether the F' factor arose by a type I or type II excision. Figure 2.19 shows that in both types of heteroduplex the F' strand carries some bacterial sequences that are missing from the F factor strand and thus form a single stranded loop unable to find a complement with which to pair. After a type II excision, the F' factor contains all of the sequences of F; and so the F strand of the heteroduplex is perfectly paired and only the F' strand bears a single strand extrusion. This thus appears as an insertion loop (also sometimes known as a deletion loop , since the same result is produced by an insertion into one strand or a deletion in the other). In a type I excision the same single strand sequence corresponding to the insertion of bacterial DNA is seen in the F' strand. But part of the F factor is left in the bacterial chromosome after a type I excision, so that the F' strand has suffered a deletion where F sequences have been replaced by the bacterial sequences gained from the chromosome; this means that the corresponding sequences in the F strand find no partner in the F' strand. A single strand loop of these sequences therefore forms on the F strand