Abstract
A cDNA library was prepared from mRNA isolated from the lepidopteran Trichoplusia ni during larval-pupal metamorphosis. Differential probing was used to identify clones for mRNAs which are suppressible by exogenous juvenile hormone treatment. In vitro transcribed cRNAs from these clones were translated in vitro and challenged with antiserum specific for a known acidic, juvenile hormone-suppressible hemolymph protein (AJSP-1) that is associated with larval metamorphosis. Three clones were found which encoded immunoreactive translation products; their identity was confirmed by comparison of the N-terminal sequence of the mature AJSP-1 protein with the cDNA sequence. As inferred from the cDNA sequence, the protein encompasses 704 amino acid residues, including a N-terminal signal peptide; widely distributed as well as more localized stronger sequence similarities indicate that the protein is distantly related to hemocyanins and hemocyanin-like insect proteins. However, on the basis of amino acid sequence and composition, immunological reactivity, and hormonal sensitivity, the protein is distinct from previously described insect proteins. Its juvenile hormone suppressibility can be ascribed to suppression of the mRNA. RNA blot analysis using the cloned cDNA as a probe demonstrated that the transcript (approximately 2.8 kilobases) is of very low abundance during the penultimate stadium but becomes very abundant during the last larval stadium, when juvenile hormone rapidly declines. Furthermore, treatment of larvae with a juvenile hormone analog strongly suppresses the abundance of the message.
Highlights
As inferred from the cDNA sequence, the protein encompasses 704 amino acid residues, including a N-terminal signal peptide; widely distributed as well as more localized stronger sequence similarities indicate that the protein is distantly related to hemocyanins and hemocyanin-like insect proteins
In our previous studies on the cabbage looper, Trichoplusia ni (Lepidoptera), we identified a number of juvenile hormonesuppressible hemolymph proteins which become very highly expressed during larval-pupal metamorphosis [14, 15]
There are a number of unrelated hemolymph proteins expressed during this developmental stage which are suppressible by JHA treatment [14]
Summary
Insects-Larvae of T. ni were reared and staged as described previously [17, 18]. Developmental age is expressed by a code which indicates the larval stage of the animal and the day since molting to that stage. CDNA Clone for Juvenile Hormone-suppressible Protein lation products were separated by sodium dodecyl sulfate-polyacrylamide gel e!ectrophoresis (SDS-PAGE). SDS-PAGE, proteins were transferred to nitrocellulose and subjected to immunoblotting as described [20], using 1:200 diluted polyclonal antiserum [16]. 66 colonies which had been shown by differential probing to possess cDNA inserts corresponding to juvenile hormonesuppressible mRNAs. The filters were prehybridized and hybridized using the cDNA insert of a positive clone identified as above through translation, immunoprecipitation, and electrophoresis of the product. Prehybridization and hybridization solutions and conditions were as described above for DNA hybridization This sequence matches the protein sequence encoded in the cDNA clone, following an 18-residue signal peptide. Dot blot analysis was performed using dots of RNA from control L5D2 and juvenile hormone analog-treated L5D2 larvae.
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