DIFFERENTIAL AND TISSUE-SPECIFIC EXPRESSION OF A GENE FAMILY FOR TYROSINE DOPA DECARBOXYLASE IN OPIUM POPPY

Two early and potential rate-limiting steps in the biosynthesis of iso quinoline alkaloids, such as morphine and codeine, in opium poppy (Pap aver somniferum) involve decarboxylation of L-tyrosine and L-dihydroxy phenylalanine (L-dopa) to yield tyramine and dopamine, respectively. A DNA fragment was amplified by polymerase chain reaction (PCR) using de generate primers designed to two highly conserved domains found in oth er aromatic amino acid decarboxylases. A poppy seedling cDNA Library w as screened with this PCR product and a cDNA (cTYDC1) for tyrosine/dop a decarboxylase (TYDC/DODC) was isolated. Two other independent cDNAs (cTYDC2 and cTYDC3) encoding TYDC/DODC were isolated by heterologous s creening with a plant tryptophan decarboxylase (TDC) cDNA as probe. A poppy genomic Library was screened with cTYDC1 and two intronless geno mic clones (gTYDC1 and gTYDC4) were isolated. The deduced amino acid s equences of all poppy clones share extensive identity with other repor ted pyridoxal phosphate-dependent decarboxylases from both plants and animals. Based on sequence homology, members of the gene family were d ivided into two subsets (cTYDC1 and gTYDC4; cTYDC2 and cTYDC3) of prot eins with predicted M(r) = 56,983 and 59,323, respectively. Within eac h subset the clones exhibit greater than 90% identity, whereas clones between subsets share less than 75% identity. Expression of gTYDC1 and cTYDC2 as beta-galactosidase fusion proteins in Escherichia coli resu lted in catalytically active enzymes immunodetectable with TDC-specifi c polyclonal antibodies. Each enzyme showed marginally higher substrat e specificity for L-dopa over L-tyrosine, but did not accept L-tryptop han and L-phenylalanine as substrates. Genomic DNA blot-hybridization analysis revealed 6 to 8 genes homologous to cTYDC1 and 4 to 6 genes h omologous to cTYDC2 in the tetraploid poppy genome. A premature transl ation stop codon was found in the gTYDC4 clone suggesting that it may not encode a functional protein. RNA blot-hybridization with probes sp ecific to the gTYDC1- or cTYDC2-like subsets showed that members of th e TYDC gene family are differentially expressed in various plant tissu es.