132 

 Discussion 



S-protein cDNA cloning 



From five possible cDNA clones isolated by antibody- 

 screening of a Lambda Zap II cDNA library from N. tabacum, 

 three identical clones encoding the bifunctional S-protein, 

 were obtained. Subcloning analysis and sequencing of the 

 entire cDNA of clone SP3 was performed. 



The other two cDNA clones, probably code for two other 

 NADP-"- dependent plant proteins, malate dehydrogenase and 

 cinnamyl-alcohol dehydrogenase. Antibody selection of these 

 two cDNA clones could be due to the presence of these 

 proteins as contaminants in the purified SDH/DQT 

 bifunctional protein. Consistent with this, both MDH and 

 CDH have very similar calculated isoelectric points (5.78 

 and 6.07, respectively) compared to the mature S-protein 

 isoelectric point of 5.96. Neither CDH nor MDH activity 

 could be detected in the purified S-protein preparation, 

 even at high protein concentration. Alternatively, MDH and 

 CDH might have common epitopes recognized by the S-protein 

 specific antibody. As a final possibility, the rabbit may 

 have developed an immune response separate from that 

 developed to the injected protein, perhaps by ingestion of 

 some plant similar to tobacco. Using the GAP analysis in 

 the GCG program, the SDH functional domain of the 

 bifunctional protein had 21% and 23% identities with CDH and 



