Gina Cassell

Two mutations that affect reproductive function

About a year ago, the students working in my laboratory and I discovered that one of the lines of Drosophila mutants that we had been studying intensely actually harbored two mutations on the same chromosome. The mutant line exhibits an abnormal reproductive behavior, the females overproduce eggs but the males fail to fertilize them. The double mutation had not been detected earlier because of the placement of the two mutated genes on the same chromosome and because the expression of the marker for one gene was epistatic to the other.

One of the mutations is approximately 2 kb 3 ' of the coding region of the gene for a previously characterized protein, Acp76A. This protein is one of at least 20 proteins that are synthesized in the accessory gland of male flies and transferred to females during copulation. As a group, the accessory gland proteins are known to affect sperm storage, female mating behavior and egg production and oviposition. The genes for a number of these proteins have been identified and sequenced and several of the proteins have been studied extensively. Acp76A is known to encode a serpin, one of a large family of proteins, most of which function as serine protease inhibitors. The exact function of Acp76A has not been identified. We have collected data that may indicate that Acp 76A plays a role in a process such as sperm storage, transfer or motility but does not affect egg production or deposition. Data collected from the mutant line before the two mutations were separated genetically indicate that, female flies mated with males from the mutant line lay 30% more unfertilized eggs than females mated with wild-type flies.

The other mutation harbored in this line lies 107bp 5 ' of candidate gene, CG7752. The gene is predicted to encode a transcription factor and it lies in an area of the chromosome that is known to encode genes for several hormone receptors and carriers. The two mutations were separated genetically. PCR-based analysis was used to show that the two mutations were separated into independent lines.

The figure above is a photograph of an agaraose gel showing the PCR products obtained when independent lines of flies resulting from the genetic separation where amplified using primers for each of the mutations. Each number represents and independent line. All of the "A"  lanes show the PCR product obtained using primers designed to amplify the Acp76A mutation. All of the "B"  lanes show the PCR product obtained using primers designed to amplify CG7752. As you can see Line   now carries the mutation for the Acp76A but not for CG7752. Line   has a mutation in CG7752 but not Acp76A. Now that the mutations are separated Gina Cassell is obtaining plasmid rescue fragments and studying the ability of the flies in each line to produce and fertilize eggs.