Cloning and Characterization of a Gene Responsible for the Lathyrus sativus Neurotoxin Degradation

Abstract

A plasmid pBYA1, capable of degrading the Lathyrus sativus neurotoxin β-N-oxalyl amino alanine (BOAA), was isolated from a soli microbe and a physical map of the plasmid constructed. A partial Sau3A library of the plasmid was then prepared in pUC18 and a recombinant clone with a 1.8 kb insert capable of growing in M9 medium, with BOAA as sole source of carbon and nitrogen, was isolated. A nested set of deletions of this 1.8 kb fragment was then generated using Bal31 and the shortened fragments subcloned in the vector pUC18. Each of these clones were sequenced by Sanger’s dideoxy method and the sequences overlapped and analysed. The 1.8 kb BOAA degrading fragment was found to contain a 630 nucleotide long coding stretch, coding for 191 amino acids. The initial 56 nucleotides were found to contain sequences resembling the Pribnow-Schaller box of prokaryotes, the “-35” sequences and a sequence resembling the “-43” region of the lac promoter.