Mapping of Smal and BamHI Restriction Sites Map of...

Mapping of Smal and BamHI Restriction Sites Map of N.otophora Chloroplast DNA

The basic strategy in constructing the SmaI and BamHI restriction sites map is to take advantage of the available information on several SmaI sites mapped on N.tabacum（Jurgenson，1980）.The SmaI restriction sites on N.otophora ct-DNA are identical to that of N.tabacum with only one exception（Kung et al.，1982）.The sequential order of SmaI fragments on the left half of the large single-copy region（see Figure 3）was arranged according to the information provided by Smal digestion of ct-DNA from several Nicotiana species（Kung et al.，1981）.This region consists of five SmaI fragments in the order（counter-clockwise）of-13-5-6-8-10-.It is 24.0 kb in length accounting for approximately 15%of the total genome.The order of Smal fragments in the other half of the large single-copy region was aligned as-3-7-1-12'-15-based on the information obtained from hybridization of SmaI and SalI fragments.Sall restriction map for N.tabacum ct-DNA of Seyer et al.（1981）was used as a standard.The SmaI restriction sites of the inverted repeats（-9-4-11-14-）（-9'-4'-11'-14'-）and the small single-copy region（-2-）were essentially adapted from the map of Jurgenson and Bourque（1980）.Thus，the complete SmaI restriction map was generated for N.otophora.Based on this map，the BamHI restriction sites were also mapped in the following manner：① primary digestion：ct-DNA was digested with SmaI and BamHI individually or in combination（see Table 1）；② secondary digestion：each SmaI or BamHI fragment was cut from low-gellingtemperature-agarose gel，melted and digested with a second enzyme BamHI or SmaI respectively；③ DNA hybridization.The sequential order of some very small BamHI fragments in a large SmaI fragment was determined by further restriction enzyme digestion using XhoI，BglI or PvuII and their known restriction sites map of N.tabacum（Seyer et al.，1981）.The ct-DNA of N.otophora is 160.5 kb in length（see Table 1）.