Vincent Gasparotto

Detecting deletions in the Hdc gene generated by Minos transposon excision in Drosophila melanogaster

Histamine is an important neurotransmitter used by photoreceptor cells and central brain neurons in the fruit fly, Drosophila melanogaster. Histamine is synthesized by the enzyme histidine decarboxylase (HDC) that is encoded by the Hdc gene. While mutants in the Hdc gene have been identified that eliminate up to 98% of Hdc activity, they do not eliminate total expression of the Hdc gene and it is possible that a stronger phenotype could be generated by deleting parts of the Hdc gene, which would result in the absence of the Hdc gene.  With the use of the Minos transposable element (Mi{ET1}) that contains the gene encoding green fluorescent protein (GFP), an excision of the Mi{ET1} element could be detected by the absence of GFP gene function.  As the (Mi{ET1} HdcMB07121) element is located in the Hdc gene's second intron, removal of the element and surrounding DNA may result in the disruption of the Hdc gene.  Once the Hdc gene is deleted, the ability to synthesize histamine will be eliminated and easily detectable when examining flies for the presence of histamine.

Currently, Minos excision experiments have yielded (306 GFP- flies out of 2044 total flies screened) for an excision rate of about 15%.  Of the 306 excision flies generated, over 80 breeding lines have been established from single excision-bearing flies having the correct genotype.  All of these lines will be examined for the lack of histamine using histamine immunocytochemical staining, which is currently under progress.  Once histamine deficient lines have been identified, the molecular extent of the deletions causing the disruption of Hdc gene function will be determined using PCR methodologies.  It is anticipated that of the 80+ GFP- breeding lines established, approximately 5% will have deletions that remove Hdc function.  These flies will then be available for phenotypic analysis in determining the function of Hdc in the central brain of Drosophila.

Faculty Mentor: Martin Burg, Biomedical Science

Page last modified August 2, 2013