Project Title: GENE EXPRESSION IN INSECT EMBRYOS

Duration: 11/13/89 - 09/30/96

Sub Class:

Integrated Control-Basic Research

Objectives: To determine the DNA sequence of ornithine decarboxylase genes from genomic DNA clones and from cDNA clones previously constructed from housefly embryonic DNA and mRNA. To elucidate the mechanisms of inhibition and activation of ornithine decarboxylase activity during embryogenesis.

Approach: The DNA sequence of housefly ODC genes and their 5' flanking regions will be determined. The Sanger method for DNA sequencing will be utilized. We also will determine if ODC 5' sequence specific transacting factors are present in embryo extracts at any time during embryogenesis. These experiments will utilize the methods of gel shift and DNA footprinting. In order to evaluate the way that ODC is attached to the membrane, we will attempt to solubilize and release an inactive form of ODC while it is still attached to components of the membrane. Several detergents and various ionic, buffer and pH conditions will be utilized in these experiments. The released ODC will then be isolated by immunoaffinity chromatography and characterized. Inactive ODC will be monitored by ODC specific antibody and Western blots.

Keywords: GENE-EXPRESSION INSECTS EMBRYOS INSECT-GENETICS DNA-SEQUENCES ORNITHINE DECARBOXYLASES ORNITHINE-DECARBOXYLASE MECHANISM-OF-ACTION DNA-CLONING GENES MUSCA-DOMESTICA DNA RNA-M ENZYME-ACTIVITY GENE-MAPPING

Progress:

Studies were carried out to characterize the ornithine decarboxylase (ODC) gene and its expression in embryos of Musca domestica. The reverse transcriptase-polymerase chain reaction (RT- PCR) was used to amplify a 900-bp fragment of the housefly gene. The RT-PCR primers were selected based on highly conserved regions of ODC proteins that had been previously deduced from DNA sequence analysis of ODC genes. The starting material for RT-PCR was polyA mRNA isolated from developing embryos at the times of ODC specific mRNA synthesis, as previously determined. The DNA product from this reaction was a 900 bp fragment, which was then cloned into M13mp18 and M13mp19 at the appropriate restriction sites. Clones containing the insert were selected and DNA sequence analysis was carried out. Comparison of the DNA sequence to known ODC sequences revealed that the RT-PCR fragment was indeed from an ODC gene. The RT-PCR product was radiolabeled and used as a probe in Northern and Southern blots. The RT-PCR product is now used to screen a cDNA clone bank that was recently constructed.
Relevance: Noncommodity Biotechnology, Biometry
Relevance: Biology of Plants and Animals
Relevance: Protection Against Insects
Relevance: Biological Cell Systems
Relevance: Invertebrates
Relevance: Biology-Molecular-Animal
General Relevance: Technology-Noncommodity
General Relevance: Invertebrates
General Relevance: Production
General Relevance: Protection

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