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CHAPTER VII 



CLONING CDNAS ENCODING THE BIFUNCTIONAL 

 S- PROTEIN, ADH AND PAT 



A cDNA library from N. tabacum was available for use in 

 cloning cDNA encoding proteins from the aromatic amino acid 

 biosynthetic pathway. Thus far, genes have not been cloned 

 for the bifunctional S-protein, arogenate dehydrogenase or 

 prephenate aminotransferase of higher plants. 



RESULTS 



Cloning aromatic pathway genes 



S-protein cDNA. Greater than 10^ PFU carrying inserts 

 from a N. tabacum library were plated with E. coli XLl-Blue. 

 Probing the library with S-protein antibody, five plaques 

 that gave blue reactions on IPTG saturated nitrocellulose 

 filters were designated SP3, SP5, SP6, SPIO, SP33 and 

 studied further. The size of the cDNA coding for the 

 bifunctional protein was expected to be about 2 kB, based on 

 molecular-weight estimations (Chapter V) and based on known 

 sequences of yeast and E. coli (Table 1-1) . Gel 

 electrophoresis of purified pBluescript containing the 

 inserted cDNAs had different mobilities on the gel (plasmid 

 DNA was a mixture of circular and supercoiled DNA) . The 



