19 



CHAPTER II 

 MATERIALS AND METHODS 



Organisms and extract preparations 

 Eukaryotes 



Nicotiana silvestris Speg. et Comes suspension cultured 

 cells originally isolated from haploid leaf material have 

 been maintained in our laboratory for about twelve years and 

 are well described physiologically and biochemically (10, 

 11, 12) . Cell populations are subcultured in stationary 

 phase (7 day) , diluted fivefold into fresh Murashige and 

 Skoog medium (62) , and grown under specified conditions in 

 controlled growth chambers (10) . For these experiments 

 (unless otherwise stated) seven-day old cells were harvested 

 by filtering through Miracloth (Calbiochem, LaJolla, CA) , 

 rinsed with nanopure water, ground in liquid nitrogen in a 

 Waring blender and stored at -70°C. until use. 



C. sorokiniana cells cultured as described by Meridith 

 and Schmidt (57) were kindly provided by Dr. Robert Schmidt. 

 Bacterial strains and plasmids 



E. coli strains used for transformation include DH5a, 

 AB1360 aroD (DQT") and SK494 aroE (SDH) . Plasmids used for 

 subcloning were pUC18, pGEMBZf (+) or pBluescript SK+ . 



