Abstract
A group of recently isolated parsley (Petroselinum crispum) cDNAs representing genes that are transcriptionally activated upon fungal infection or elicitor treatment have been demonstrated to encode tyrosine decarboxylase (TyrDC). The deduced TyrDC protein sequence shares extensive similarity with two functionally related enzymes, tryptophan decarboxylase from periwinkle and dopa decarboxylase from Drosophila melanogaster. Expression of TyrDC cDNA in Escherichia coli yielded catalytically active protein with high substrate specificity for tyrosine. All four identified parsley TyrDC genes have been cloned and encode at least three TyrDC isozymes. Treatment of cultured parsley cells with fungal elicitor caused very rapid and transient increases in TyrDC mRNA and enzyme activity levels.
Highlights
A group of recently isolated parsley (Petroselinum crispum)cDNAs representing genes that are transcriptionally activated upon fungal infection or elicitor treatment have been demonstrated to encode tyrosine decarboxylase (TyrDC)
Investigations of TyrDC have been hampered by the lack of suitable antibodies or DNA probes
Ten pg of DNA was digested with appropriate endonucleases, clones were isolated from a parsley cDNAlibraryusinga separated on 0.8% agarose gels, and transferred to Hybond-N nylon novel differential screening approach(Somssich et al, 1989)
Summary
Sequence Analysis of E L I 5 cDNAs-Recently, 62 cDNA (1980). Ten pg of DNA was digested with appropriate endonucleases, clones were isolated from a parsley cDNAlibraryusinga separated on 0.8% agarose gels, and transferred to Hybond-N nylon novel differential screening approach(Somssich et al, 1989). Expression of E L I 5 cDNA in E. coli-Expression of protein encoded by the ELI 5 cDNAs in E. coli was achieved using a modified version of the prokaryoticexpression vector, pGEX-2T (modpGEX) These clones represented 18distinct gene families (designated PR1,PR2,andELI 3-19) whose transcriptionalactivities increased rapidly and transiently upon fungal elicitor treatment of cultured parsley cells. Sequence analysis of the longest available cDNA from the ELI 5 gene family (1.4 kb), followed by comparison of the deduced amino acid sequence to proteins in the SWISS database, revealedlarge similarity to known aromaticamino acid decarboxylases. One 1.68-kb cDNA, designated TyrDC-2, Following adsorption, theagarose beads were centrifuged, the super- contained one open-reading frameof 514 amino acids with a natant removed and thebeads washed four times with N E T P buffer and once with TC P buffer (50 mM Tris-HC1, p H 8.0, 150 mM NaC1, 2.5 mM CaC12, 0.1% p-mercaptoethanol,and 25 pM pyridoxal 1phosphate). TheparsleyTyrDC cDNAsEncodeTyrosine Decarboxylases-To determinethesubstrate specificityof TyrDC, TyrDC-2 and TyrDC-3 were cloned into a modified version
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