Cloning and sequence analysis of cDNA encoding a putative juvenile hormone esterase from the Colorado potato beetle.
In the Colorado potato beetle, Leptinotarsa decemlineata, reproduction and diapause are mediated by the juvenile hormone (JH) titer in the hemolymph. This titer is controlled by JH synthesis in the corpora allata and by JH degradation. The main pathway of JH degradation is by JH esterase in the hemolymph. The native JH esterase appeared to be a dimer consisting of two 57 kDa subunits (Vermunt et al., 1997). The 57 kDa subunit of JH esterase was digested with endoproteinase Lys-C and the digestion products were separated by reversed phase HPLC. Three different peptides were collected and sequenced. The amino acid sequence of one peptide showed high similarity to fragments of other insect esterases. Based on the amino acid sequence of these peptides, degenerate primers were constructed for RT-PCR. A PCR product of 1.3 kb was obtained and sequenced. This product was used to screen a cDNA library for a complete cDNA copy and to analyze the messenger RNA from larvae and adult beetles. The size of the messenger RNA was 1.7 kb. The complete amino acid sequence of the protein was deduced from the nucleotide sequence of overlapping clones from a cDNA library and a 5'RACE product. An open reading frame (ORF) of 1545 base pairs encoded a 57 kDa protein with a predicted pI of 5.5. The ORF contained the sequence of the three peptides. It showed no significant homology to other proteins present in databases, but it did contain several functional esterase motifs.
Vermunt
A M
AM
Department of Entomology, Wageningen Agricultural University, The Netherlands. Adriaan.Vermunt@medew.ento.wau.nl
Koopmanschap
A B
AB
Vlak
J M
JM
de Kort
C A
CA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular; D001517:Coleoptera / Q000201:enzymology* / Q000235:genetics; D003120:Colorado; D018076:DNA, Complementary; D019281:Dimerization; D015723:Gene Library; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000469:parasitology
D018076:DNA, Complementary; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C038003:juvenile hormone esterase