Abstract
A physical technique known as two-dimensional S1 nuclease heteroduplex mapping has been applied to genomic DNA from the Gram-negative coccus Neisseria gonorrhoeae. This has resulted in the detection of two novel types of repetitive sequences. The first type is a repetitive sequence family of 152 base pairs (bp), whose ends are composed of inverted repeats of 26 bp. There are approximately 20 copies of this sequence, in both N. gonorrhoeae and Neisseria meningitidis (Correia, F., Inouye, S., and Inouye, M. (1986) J. Bacteriol. 167, 1009-1011). The second type of sequence is a 1443-bp duplication in the N. gonorrhoeae genome. The two classes of sequence are linked positionally. Each copy of the long duplicated sequence is adjacent to a member of the 152-bp repetitive sequence. In one instance two copies of the 152-bp repetitive sequence are separated by a 436-bp central region and are in an inverted orientation with respect to one another, resembling a compound transposable element.
Highlights
A physical technique known as two-dimensional S1 elements were found at the end of each copy of a 1443-bp nuclease heteroduplex mapping has been applied to genomic DNA from the Gram-negative coccus Neisseria gonorrhoeae
In one instance two copies of the 152-bp repetitive sequence are separated by a 436-bp central region and are in an inverted orientation with respect to one another, resembling a compound transposable described by Maniatis et al (7)
The actual construction of pNG273 and pNG293 is described in Correia et al (5)
Summary
Two-dimensional SI Nuclease Heteroduplex Mapping with pNG293 and pNG273“We applied the two-dimensional S1 nuclease heteroduplex mapping (2DS1) technique to AluIdigested genomic DNA from N. gonorrhoeae (5). The heteroduplexes appear, on the second-dimension gel, as discrete spots of ethidium bromide-stained DNA, which migrate faster than a prominent diagonal of homoduplex DNA. Kb on the second-dimension gel, hybridized to two fragments from the Hind[111] genomic blot. These fragments of 11.0 and 3.6 kb were cloned into pUC9 and designated as pNG293 and pNG273, respectively. C c pNG293 and pNG273 were run as controls (panels a and c, Fig. l ) , along with the heterorenaturation (shownby an arrow in part b, Fig. 1).The heterorenaturation gel shows a distinct spot that migrates at 1.1kb in the second dimension (shown by an arrow in part b, Fig. 1).The fragments on the diagonal pNG294. The fact that we were able to reproduce spot H using pNG293 and pNG273 demonstrates that these two plasmids carry two independent chromosomal regions, which respectively contain 1.1-kb common regions
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