Molecular Analysis of acDNA and Gene Encoding a Protein From Rigid Cuticles of the Giant Silkmoth, Hyalophora Cecropia
Lampe, David Joseph
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https://hdl.handle.net/2142/72505
Description
Title
Molecular Analysis of acDNA and Gene Encoding a Protein From Rigid Cuticles of the Giant Silkmoth, Hyalophora Cecropia
Author(s)
Lampe, David Joseph
Issue Date
1992
Doctoral Committee Chair(s)
Willis, John S.
Department of Study
Entomology
Discipline
Entomology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Biology, Entomology
Abstract
HCCP66 is an abundant protein of rigid cuticles of the larva and pupa of Hyalophora cecropia. It is secreted by a persistent population of epidermal cells with a dramatically different spatial distribution between the larval and pupal stages. There is reason to believe that its expression is regulated by juvenoids and ecdysteroids, making this an attractive model system in which to study metamorphosis at the genetic level.
Using reverse transcription-PCR, it was possible to isolate a partial cDNA for HCCP66 from pupal epidermis. This product was used in turn to isolate a more complete cDNA from a cDNA library from pupal wing epidermis and, subsequently, a genomic clone. Conceptual translation of the cDNA showed the protein to be small and acidic. Northern analyses were consistent with data obtained for the distribution of the protein in cuticle.
Hydrophilic regions of many cuticle proteins were found to have similar sequences, although complete sequences were highly divergent at their termini. Surprisingly, the regions of similarity were shared with a group of proteins from flexible cuticles previously thought to belong to a distinct protein family. Analyses made it possible to postulate that most of the sequenced cuticle proteins, as well as those isolated from chorion and oothecae, can be united into an insect structural protein superfamily.
Sequencing showed the gene to contain one large intron which interrupted the coding sequence of the signal peptide, a feature typical for cuticle protein genes. Nearly half the length of the intron was composed of 3 imperfect tandem repeats of an abundant Cecropia sequence. Sequence comparisons indicated that the inserted element was a SINE type of mobile DNA.
Sequences were found in the promoter region of the gene which matched an Octamer (Oct) sequence and resembled a Drosophila ecdysone response element (EcRE). Gel retardation assays revealed that there were proteins present in cell extracts from different tissues and stages that specifically bound the Oct sequence, but no binding could be detected to the putative EcRE.
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