41 

 until use. Controls were prepared by streaking auxotrophic 

 mutant strains on M9 medium plates +/- aromatic amino acids. 

 Transformation of plasmid DNA 



Either pUC18, pGEM5zf(+), or pBluescript SK+ plasmids 

 were incubated with competent cells of DH5q; on ice, heated 

 to 42°C for 90 sec, cooled in an ice bath before addition of 

 SOC medium (68) . The cells were then incubated at SV^C for 

 1 h before spreading on LB-AMP plates. A colony from these 

 transformed cells was streaked onto a fresh LB-AMP plate. 

 Controls included spreading of competent cells without 

 plasmid or plasmid without insert onto LB-AMP plates. 

 Functional complementation 



E. coli mutant strains, aroD (DQT") and aroE (SDH") , 

 were prepared for transformation with the pBluescript 

 plasmids carrying cDNA clones and were plated on LB-AMP 

 plates. After growth, colonies were streaked onto M9 

 plates. Controls included nontransformed competent cells or 

 plasmid spread on LB-AMP plates, and competent mutant 

 strains spread on M9 plates +/- aromatic amino acids. 

 Initial sequencing of cloned cDNA 



Purified pBluescript plasmid with cloned inserts were 

 submitted to the DNA Sequencing Core Facility, ICBR, UF, for 

 initial 5' and 3' end sequencing (from the T3 and T7 

 promoter sites) . 



