The long term objective of this study is to understand the basic mechanism of crown-gall tumor formation. Since understanding of the molecular basis of crown-gall tumor formation requires information on the gene products of T-DNA and their functions the following topics will be investigated: 1. To purify and identify an unusual acidic compound synthesized by a heretofore unknown synthase in octopine type tumors. We have obtained evidence that octopine type crown-gall tumors accumulate N2-(l-carboxyethyl)-L-glutamine and a portion of the accumulated N2-(l-carboxyethyl)-L-glutamine is synthesized by a TR-DNA encoded synthase not associated with agropine synthesis. The putative N2-(l-carboxyethyl)-L-glutamine synthesized by the unknown synthase will be isolated from an octopine minus mutant tumor (S-351 tumor). The structure of the purified compound will be determined by mass and NMR spectroscopies. The stereochemistry of this compound and its opine nature will also be investigated. 2. To isolate and characterize the new synthase. The new synthase will be purified from S-351 tumor tissue and l5955/l tumor line both of which lack octopine synthase. Properties of the synthase such as substrate specificity, Km, pH optimum and molecular and subunit weight will be studied. 3. To identify the T-DNA gene for the new synthase. Our preliminary studies indicate that the new synthase gene is one of the two left most genes (4' and 5') of TR-DNA. The exact location of the new synthase gene will be determined by: (1) analyzing the putative N2-(l-carboxyethyl)-L-glutamine in tumors with multiple mutations; (2) assaying the new synthase in tumors with either both genes 4' and 5' deleted or only gene 4' inserted. The information obtained from these studies should also serve to clarify some of the problems regarding the mechanism of human tumor formation.
NIH ExPorter
NIH ExPorter