Molecular characterization of a blood-meal induced trypsin from the mosquito Aedes aegypti.

Persistent Link:
http://hdl.handle.net/10150/185803
Title:
Molecular characterization of a blood-meal induced trypsin from the mosquito Aedes aegypti.
Author:
Barillas Mury, Carolina Veronica.
Issue Date:
1992
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Adult and laIval midgut trypsins from the mosquito Aedes aegypli were isolated using benzamidine affinity chromatography. The cDNA of the late trypsin induced by the blood meal was isolated using an anti-trypsin monoclonal antibody, cloned and sequenced. The 862 bp sequence codes for a 257 amino acid protein, which is presumably a trypsin precursor, since the sequence of purified mosquito trypsin begins at residue 26, immediately following an arginine residue in the precursor. The amino terminal 25 amino acids in the precursor are composed of a putative 15 amino acid signal peptide and a 10 amino acid activation peptide. The activation peptide in the mosquito is different from that of vertebrate trypsinogens and suggests that activation takes place by tryptic cleavage. The deduced amino acid sequence is homologous to that of other trypsins in those residues around the catalytic triad, and in several residues which are found only in trypsins. However, the sequence of the specificity pocket in mosquito trypsin, KESPC, differs from that found in other trypsins, KDSC. The Asp is thought to bind the basic residue of the substrate, and the Glu in the mosquito trypsin may serve the same role. The changes in trypsin protein and mRNA levels following a blood meal indicate that an important component of the regulation of trypsin synthesis is at the transcriptional level. A genomic clone of "late trypsin" was isolated, mapped, and 1.2 kb of the upstream regulatory region were sequenced. The gene has no introns within the coding region. A TAT A box consensus sequence (TAT AAA) was found at position -31 from the 5' end of the mature mRNA. A cluster of five repeat sequences homologous to the GCN4 DNA binding site was found within 200 nucleotides upstream of the cap site. GCN4 is required for derepression mediated control of general amino acid biosynthesis in response to amino acid starvation in yeast. This suggests that a similar protein might regulate expression of the late trypsin gene in the mosquito. Southern blot analysis of genomic DNA suggests that the regions flanking the late trypsin coding region are polymorphic.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Dissertations, Academic.; Biochemistry.; Aedes aegypti.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Biochemistry; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Wells, Michael

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleMolecular characterization of a blood-meal induced trypsin from the mosquito Aedes aegypti.en_US
dc.creatorBarillas Mury, Carolina Veronica.en_US
dc.contributor.authorBarillas Mury, Carolina Veronica.en_US
dc.date.issued1992en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractAdult and laIval midgut trypsins from the mosquito Aedes aegypli were isolated using benzamidine affinity chromatography. The cDNA of the late trypsin induced by the blood meal was isolated using an anti-trypsin monoclonal antibody, cloned and sequenced. The 862 bp sequence codes for a 257 amino acid protein, which is presumably a trypsin precursor, since the sequence of purified mosquito trypsin begins at residue 26, immediately following an arginine residue in the precursor. The amino terminal 25 amino acids in the precursor are composed of a putative 15 amino acid signal peptide and a 10 amino acid activation peptide. The activation peptide in the mosquito is different from that of vertebrate trypsinogens and suggests that activation takes place by tryptic cleavage. The deduced amino acid sequence is homologous to that of other trypsins in those residues around the catalytic triad, and in several residues which are found only in trypsins. However, the sequence of the specificity pocket in mosquito trypsin, KESPC, differs from that found in other trypsins, KDSC. The Asp is thought to bind the basic residue of the substrate, and the Glu in the mosquito trypsin may serve the same role. The changes in trypsin protein and mRNA levels following a blood meal indicate that an important component of the regulation of trypsin synthesis is at the transcriptional level. A genomic clone of "late trypsin" was isolated, mapped, and 1.2 kb of the upstream regulatory region were sequenced. The gene has no introns within the coding region. A TAT A box consensus sequence (TAT AAA) was found at position -31 from the 5' end of the mature mRNA. A cluster of five repeat sequences homologous to the GCN4 DNA binding site was found within 200 nucleotides upstream of the cap site. GCN4 is required for derepression mediated control of general amino acid biosynthesis in response to amino acid starvation in yeast. This suggests that a similar protein might regulate expression of the late trypsin gene in the mosquito. Southern blot analysis of genomic DNA suggests that the regions flanking the late trypsin coding region are polymorphic.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectDissertations, Academic.en_US
dc.subjectBiochemistry.en_US
dc.subjectAedes aegypti.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineBiochemistryen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorWells, Michaelen_US
dc.contributor.committeememberHaussler, Marken_US
dc.contributor.committeememberLaw, Johnen_US
dc.identifier.proquest9225166en_US
dc.identifier.oclc712303076en_US
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