MOLECULAR-CLONING OF CDNAS AND GENES-CODING FOR BETA-METHYLCROTONYL-COA CARBOXYLASE OF TOMATO

Tomato cDNA and genomic clones were isolated by using as a probe a cDN A clone that had originally been identified by its ability to direct t he synthesis of a biotin-containing polypeptide in Escherichia coli. T he nucleotide sequences of the newly isolated cDNAs indicate that they are clones of a single mRNA molecule. However, one of the cDNA clones contains an insertion of a sequence which we identified as an unsplic ed intron. The amino acid sequence deduced from the nucleotide sequenc e of the cDNAs showed similarity to regions of previously sequenced bi otin enzymes, indicating that the isolated cDNAs code for a biotin-con taining protein. Portions of the cDNAs were expressed in E. coli as gl utathione S-transferase or beta-galactosidase fusion proteins. Each fu sion protein was purified and used to immunize rabbits. The resulting antisera recognized a 78-kDa biotin containing polypeptide in tomato l eaf extracts. In addition, both antisera specifically inhibited beta-m ethylcrotonyl-CoA carboxylase activity in extracts from tomato leaves. These characterizations have identified the isolated tomato cDNAs and genes as coding for the 78-kDa biotin subunit of beta-methylcrotonyl- CoA carboxylase. Comparison of the deduced amino acid sequence of the biotin subunit of beta-methylcrotonyl-CoA carboxylase with other bioti n enzymes suggest that this subunit contains the biotin carboxylase an d biotin carboxyl carrier domains.