Characterization of a cytosolic juvenile hormone binding protein from the house fly, Musca domestica L. and its relationship to metabolic resistance to insecticides /
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| Other Authors: | , |
| Format: | Thesis Book |
| Language: | English |
| Published: |
1991.
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| Online Access: | Link to OAKTrust copy |
| Abstract: | Metabolic resistance to insecticides has been most extensively studied in the house fly, Musca domestica L. Biochemical and genetic studies suggested that cytosolic juvenile hormone binding proteins (JHBPs) were associated with metabolic resistance (Carino, 1987). The involvement of cytosolic JHBPs in insecticide metabolism and resistance has also been reported in other insects such as in Heliothis zea (Muehleisen et al., 1989, 1990) and Drosophila melanogaster (Shemshedini and Wilson, 1990). In this dissertation, a protocol for identification, isolation, purification, characterization, and cloning of a cytosolic JHBP from fat body cytosol of third instar house fly larvae is documented. This protocol was composed of several critical steps as follows: identification of a cytosolic JHBP by photolysis; purification by differential centrifugation, ammonium sulfate precipitation, dialysis, sucrose density gradient centrifugation, and reversed-phase high-performance liquid chromatography (HPLC); elucidation of the amino terminal amino acid sequence by trichloroacetic acid (TCA) precipitation and Edman degradation; and cloning by reverse transcription and polymerase chain reaction (PCR) using mRNAs prepared from the same tissue. Biochemical measurements indicated that the cytosolic JHBP had a Kd of 8 to 9 nM and existed as a dimer. This protein had a molecular weight of approximately 22 kDa (44 kDa as a dimer) and an amino terminal amino acid sequence of NH$ sb3$-Asp-Ala-Glu-Ile-Val-Lys-Ile-Val-Lys-Gln-Val-Ser. Its levels were highest in third instar larvae and gradually declined in later developmental stages. The presence of this cytosolic JHBP was synchronous with high titers of both JH and cytochrome P450VIA1. This pattern was similar in both resistant and susceptible strains. However, both the cytosolic JHBP and cytochrome P450VIA1 were overproduced in a resistant strain. A confirmation test for the cloned 1.5 kbp DNA sequence by Northern dot blot with cytosolic RNA preparations from house fly egg, larva, and pupa was not successful. While this failure may indicate that the gene cloning was not successful, it more likely reflects a low copy number of the target mRNA. |
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| Item Description: | Typescript (photocopy). Vita. "Major subject: Toxicology." |
| Physical Description: | x, 97 leaves : illustrations ; 29 cm |
| Bibliography: | Includes bibliographical references. |