125 

 left) indicated that Hindi II, Banll and EcoRV had at least 

 several restriction sites within the cDNA insert, and that 

 SphI and Ndel, each had one restriction site located within 

 the insert. The remaining restriction enzymes shown in Fig, 7- 

 IB, only had one site within the multiple cloning region of 

 the plasmid. From this information and from the known RE 

 sites of the 3' and 5' sequenced ends of the cDNA clone 

 (determined by GCG Map analysis) a strategy for subcloning was 

 begun and continued as information about RE sites was obtained 

 through subcloning (Fig. 7-3) . 



After appropriate RE were used to obtain fragments of 

 suitable size for sequencing, the bands were excised from 

 agarose gels (Fig. 7-4A) and prepared for transformation. 



To confirm that the transformed cells carried plasmids 

 containing the appropriate fragments, a cracking procedure 

 (Promega protocol, Madison, WI) , followed by an agarose gel 

 was performed (Fig. 7-4B) . This gel showed that the plasmids 

 contained inserts of the correct size and were ready to be 

 purified for sequencing. Agarose gels containing the purified 

 plasmid DNA carrying the various fragment sizes are shown in 

 Fig. 7-4C. 



After sequencing the cDNA fragments on an available Li- 

 Cor sequencer, overlapping areas were placed together to yield 

 a complete cDNA sequence. For correction of sequencing 

 errors, multiple sequencing of all fragments was done in both 

 directions. The complete nucleotide sequence and the deduced 



