Research Interests

In my laboratory we use the fruitfly, Drosophila melanogaster, with the powerful molecular genetic techniques associated with this model organism to isolate and study novel genes. My main interest is in genes that are important in the development of tissue that are derived from mesoderm, in particular, muscle. Sometimes, however, the research itself leads you in other directions. Consequently, we also are working two separate genes whose function affects the reproductive system.

As an outgrowth of the work on the reproductive system, I have also become interested in a number of genes that code for proteins that have their primary function in some physiological system besides the reproductive system, but affect reproductive behavior indirectly. We are specifically interested in the gene mutation that leads to white eyes and that which leads to yellow body color. It has been known for some time that males flies with these mutations also exhibit defective reproductive behavior. Despite this, fecundity in these mutant lines is normal. It appears that females in these mutant lines have adapted and mate readily with these males when wiuld type females will not. We are interested in the source of the adaptation.

Many of the projects in my laboratory start with the use Enhancer trap techniques in Drosophila melanogaster. Enhancer trap analysis is a set of gene mutation and analysis techniques that were developed specifically for use in Drosophila. The techniques are based on the insertion of a P-transposable element (P-element) in Drosophila. Transposable elements are short pieces of DNA that can insert and remove themselves from the chromosomes. The P-element, therefore, are capable of causing insertional mutations. At the same time, they provide tools for the analysis of the mutated gene. Using these tools we can easily determine the protein expression pattern of the gene and the location of the gene on Drosophila chromosomes. The P-element also contains a cloning vector, which allows us to obtain a genomic clone in the vicinity of the mutated gene. After isolation of this initial clone, we use standard molecular genetic techniques in combination with techniques in cell biology and physiology to study the structure and function of the mutated gene.

Flight Muscle Genes

One of the ongoing projects in my laboratory involves the study of a flight-impaired mutant that was created by P-element insertion. Flies in this mutant line are able to fly normally on eclosion but gradually lose the ability to fly over the next few weeks. The gene that is affected by the mutation appears to be expressed in all early mesoderm but is restricted to flight muscles in the adult. Electron microscopy of the flight muscle in these mutants reveals severely disrupted tissue morphology.

The Drosophila genome that has now been completely sequenced. by the Berkeley Drosophila Genome Project. We obtained a clone of the segment of DNA that has been mutated in this line and had it sequenced. The cloned DNA was found to lie within a segment of the chromosome that is not likely to contain the coding region of a gene, but which is probably the regulatory region for the gene which is disrupted..

A graduate student in my laboratory is working on studies designed to identify the gene affected by the P-element insertion. understand the regulation and function of the gene. She is using RT-PCR to compare the level of expression of a number of candidate genes in wild type and mutant flies. She will also look at the temporal and spatial expression pattern of the gene using standard Northern analysis. Graduate Student Research

Reproductive Mutants

During the Fall 2000 semester, the students working in my laboratory and I discovered that the a P-element line of flies that we had been working with actually harbored two mutations on the same chromosomal. This 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 3Õ 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. This data was presented at the Annual Drosophila Research Conference in Washington, D.C. in March 2001. The other mutation harbored in this line lies 107bp 5Õ 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. In the summer 2001, genetic manipulation of both mutants lines were undertaken in an attempt to cause mutation directly in the coding regions of these two genes. We are in the process of using a PCR-based screen for mutations within the coding sequence of one of both of the presumably affected genes.

Temperature-Dependent Sex Determination in Turtles

While sex determination in most species is genetically determined, in many species of egg-laying amniotes sex is determined by incubation temperature. Typically, lower incubation temperatures result in male hatchlings and higher incubation temperatures result in female-hatchlings. This phenomenon has been studied extensively in alligators and several species of turtles and is important for conservation purposes as well as for a thorough understanding of the process of sex determination itself. During spring/summer 2002, Dr. Russell Burke and I will be completing a project designed to determine the temperature-sensitive period in Malaclemys terrapin, a temperature-sensitive species found locally. While this descriptive information is important to Dr. BurkeÕs work in conservation biology, as a developmental biologist I would like to understand the mechanism that underlies this process of temperature-dependent sex determination. This summer (2002) students in my laboratory will be testing a hypothesis that the temperature sensitive switch that determines sex in turtles is a change in pH with temperature. We will be measuring the pH and carbon dioxide levels of yolk and albumin in eggs incubated at differnt temperatures. We will also measure the pH sensitivity of key enzymes that transform a common precursor into androgens and estrogens.