Cloning, characterization, and developmental expression of a putative farnesoic acid O-methyl transferase in the female edible crab Cancer pagurus.
Farnesoic acid methyl transferase (FAMTase) catalyzes methylation of farnesoic acid to yield the crustacean juvenoid, methyl farnesoate (MF). A full-length cDNA encoding a 275 amino acid putative FAMTase has been isolated from the mandibular organ of the female edible crab (Cancer pagurus) by reverse transcriptase-polymerase chain reaction in conjunction with cDNA library screening. A high degree of sequence identity was found between this and other putative crustacean FAMTases. Conceptual translation and protein sequence analysis suggested that phosphorylation could occur at multiple sites in the FAMTase. This finding is consistent with the recent observation that endogenous FAMTase activity in mandibular organ extracts can be regulated by phosphorylation in vitro. We demonstrated that the recombinant FAMTase could be expressed as a LacZ-fusion protein in Escherichia coli and have undertaken its partial purification from inclusion bodies. In an established assay system, the recombinant FAMTase lacked activity. Northern blotting demonstrated widespread expression of an approximately 1250-nucleotide FAMTase transcript in female C. pagurus tissues. Levels of FAMTase transcripts in mandibular organs of female C. pagurus were found to fluctuate during vitellogenesis and embryonic development. Throughout the spring of 2002, an HPLC-based method was used to measure hemolymph MF titers in more than 70 female specimens of C. pagurus, which segregated into "high MF" and "low MF" groups. The high MF titers, which occurred before or during early vitellogenesis, coincided with, or were preceded by, elevated levels of putative FAMTase mRNA in the mandibular organs.
Ruddell
Carolyn J
CJ
School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, United Kingdom. reeshh@liv.ac.uk
Wainwright
Geoffrey
G
Geffen
Audrey
A
White
Michael R H
MR
Webster
Simon G
SG
Rees
Huw H
HH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D015152:Blotting, Northern; D003386:Brachyura / Q000201:enzymology* / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005231:Fatty Acids, Unsaturated / Q000097:blood; D005260:Female; D008780:Methyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D006113:United Kingdom
D017931:DNA Primers; D018076:DNA, Complementary; D005231:Fatty Acids, Unsaturated; C032429:methyl farnesoate; D008780:Methyltransferases; C032433:farnesoic acid O-methyltransferase