Characterization of the dopa decarboxylase gene of Manduca sexta and its suppression by 20-hydroxyecdysone.
Manduca dopa decarboxylase (DDC) cDNA was isolated, sequenced, and found to be most closely related to Drosophila DDC (72% amino acid identity). Culture of Day 2 fourth instar larval epidermis with 20-hydroxyecdysone (20E) showed that 20E was necessary to determine the later expression of the gene, but its removal was required for this expression to occur. Experiments with the protein synthesis inhibitors, cycloheximide and anisomycin, and the mRNA synthesis inhibitor, alpha-amanitin, showed that 20E induced a protein(s) which suppressed transcription of the DDC gene. Gel mobility shift assays using epidermal extracts and various fragments of the first 1.1 kb of the 5' flanking region of the DDC gene showed only one DNA fragment 87 to 167 bp upstream of the 5' initiation site that bound a nuclear protein(s) with the expected developmental specificity. The protein was abundant at the time of high ecdysteroid titer when no DDC mRNA was present, but low both before the rise (no DDC mRNA) and after the decline of ecdysteroid titer (maximal DDC mRNA). Thus, this protein(s) is a candidate for an ecdysteroid-induced transcription factor which acts to suppress DDC transcription. DNase I footprinting assays confirmed by use of a specific oligonucleotide showed that this protein(s) bound to the sequence 5'-GGCTTATGCGCTGCA-3'.
Hiruma
K
K
Department of Zoology, University of Washington, Seattle 98195, USA.
Carter
M S
MS
Riddiford
L M
LM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism; D004296:Dopa Decarboxylase / Q000235:genetics*; D004441:Ecdysterone / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D015723:Gene Library; D017344:Genes, Insect / Q000235:genetics*; D018402:Manduca / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D004441:Ecdysterone; D004296:Dopa Decarboxylase