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GelReader 2.0 shareware package for the Macintosh (NCSA Software Tool

Group at the Center for Prokaryotic Genome Analysis, University of Illinois).

 Mapping Mspl and Ddel sites. Smaller regions of 178 were purified from

SeaPlaque. These regions were digested with Mspl and Ddel in the presence

and absence of other restriction enzymes known to have recognition sites

within the region. One-half of each digest was electrophoresed in 3% agarose

and visualized by staining with ethidium bromide; the other half was end-

labeled (Sambrook, Fritsch & Maniatis 1989), electrophoresed in agarose, and

visualized by autoradiography. Fragment sizes were estimated using the

GelReader program.

 The correspondence of the fragments composing the Mspl and Ddel

variants to the physical map was determined by probing Southern blots with

smaller regions of 178. The same regions of 178 used for mapping the Mspl

and Ddel sites were sequentially radiolabeled by random-priming (Feinberg &

Vogelstein 1983, 1984) and hybridized to the blots shown in Figures 2, 3, and

5-7.

 Subcloning pB178. Most of the 9.45kb insert was subcloned into the

plasmid vector pGEM3Z (Promega). The subclones were given the prefix

pG178, and each was identified by a suffix specific for the region subcloned

(see Figure 12). The pG178 subclones were replicated and purified in the same

manner as pB178. The subcloned inserts were excised and extracted from

SeaPlaque. The ends of the subclones were sequenced, using the M 13 forward