Kenneth C. Burtis - US grants

Sex determination in Drosophila is controlled by a regulatory hierarchy that has been well characterized at both the genetic and molecular levels. However, as with other regulatory gene networks that participate in the control of Drosophila development, very little is known about either the identify of the genes actually involved in the terminal steps of differentiation (sometimes referred to as the "downstream genes") or about how these terminal genes are controlled by the regulatory genes immediately above them in the hierarchy. The objectives of the work set forth in this proposal are to identify as many as possible of the downstream genes responsible for the differentiation of sexually dimorphic adult tissues in Drosophila, and to investigate the molecular mechanisms by which doublesex, the bottom regulatory gene in the somatic sex determination hierarchy, controls the expression of these downstream genes. We will approach these objectives in two ways; by isolating genes specifically expressed in the male and female genital discs, using sensitive novel cDNA subtractive hybridization protocols, and by studying the interaction of the doublesex proteins with yolk protein genes as a model system for the control of downstream gene expression; thus yielding new insights into molecular mechanisms controlling differentiation. It has been known since the 1920s that the determination of sex in the fruit fly Drosophila melanogaster is controlled by the ratio of the number of X chromosomes to the number of sets of autosomes in an individual. When this ratio is 0.5 (as in an XY individual), the individual is male; when the ratio is 1.0 (as in an XX individual) the individual is female. It has not been until the last decade that the genetic pathway and some of the molecular details of this sex determination mechanism have been worked out. It is now clear that the process of choice whether to develop as a male or as a female is under the control of a small number of genes which interact with one another in a complex fashion. The author of this proposal has made previous contributions to our understanding of these interactions. As yet we know very little about how the choice of sexual pathway is implemented. This is a proposal to investigate this unexplored area.

The primary goal of this proposal is to clone the Fanconi anemia A (FA-A) gene of man through an analysis of the functionally homologous mus3O8 gene of Drosophila. This hematological disorder is a candidate for human gene therapy. Current support for gene homology between these two organisms is provided by the observation that among several mutagen sensitive disorders in man only Fanconi anemia is characterized by hypersensitivity to DNA cross-linking agents and concurrent insensitivity to monofunctional alkylating agents. An extensive search for analogous mutants in Drosophila has revealed only one autosomal gene with this property; the mus3O8 gene. The presence of this unique spectrum of mutagen sensitivity in mutants of only one disorder in man and one in Drosophila strongly suggests that the mus3O8 gene encodes a function analogous to one of the Fanconi genes. That suggestion is further supported by the demonstration that both Fanconi anemia cells and mus308 cells exhibit an elevated frequency of spontaneous chromosomal aberrations. Two additional rare phenotypes are common to both the mus3O8 mutants and cells deficient in the FA-A gene. These are a failure to recover DNA synthesis following mutagen treatment and the unique alteration of a deoxyribonuclease. Because the mus3O8 mutants and FA-A cells share a total of four rare properties, there is therefore a strong probability that they are functionally related. This potential functional homology has stimulated efforts to clone the Drosophila mus3O8 gene in preparation for cloning the human analogue. The feasibility of that approach is supported by numerous examples in which cloned genes from one of these organisms has been employed to recover the homologous gene from the alternate organism. Recovery of the FA-A gene can be definitively verified by testing the capacity of the cloned gene to complement the hypersensitivity of FA-A cells to DNA crosslinking agents following transfection. This approach has been chosen because all of the direct mammalian efforts, including an approach recently employed to clone the FA C gene, have thus far failed to recover the FA-A gene. Drosophila was chosen for this study because gene cloning in this organism is facilitated by genetic analysis, the presence of giant polytene chromosomes and a small genome size. It is anticipated that recovery of the human gene will place our collaborators in a position to determine if it can be employed to reverse the lethal effects of Fanconi anemia through gene therapy. This approach is particularly pertinent to Fanconi anemia because the most frequent cause of lethality in this disease is a failure of bone marrow cells to proliferate in late adolescence. This disorder can be reversed in Fanconi children, however, by bone marrow transplantation when a compatible donor is available. If, however, the patients own stem cells could be provided with the normal gene, then each individual could serve as his or her own donor.

The primary goal of this proposal is to clone the Fanconi anemia A (FA-A) gene of man through an analysis of the functionally homologous mus3O8 gene of Drosophila. This hematological disorder is a candidate for human gene therapy. Current support for gene homology between these two organisms is provided by the observation that among several mutagen sensitive disorders in man only Fanconi anemia is characterized by hypersensitivity to DNA cross-linking agents and concurrent insensitivity to monofunctional alkylating agents. An extensive search for analogous mutants in Drosophila has revealed only one autosomal gene with this property; the mus3O8 gene. The presence of this unique spectrum of mutagen sensitivity in mutants of only one disorder in man and one in Drosophila strongly suggests that the mus3O8 gene encodes a function analogous to one of the Fanconi genes. That suggestion is further supported by the demonstration that both Fanconi anemia cells and mus308 cells exhibit an elevated frequency of spontaneous chromosomal aberrations. Two additional rare phenotypes are common to both the mus3O8 mutants and cells deficient in the FA-A gene. These are a failure to recover DNA synthesis following mutagen treatment and the unique alteration of a deoxyribonuclease. Because the mus3O8 mutants and FA-A cells share a total of four rare properties, there is therefore a strong probability that they are functionally related. This potential functional homology has stimulated efforts to clone the Drosophila mus3O8 gene in preparation for cloning the human analogue. The feasibility of that approach is supported by numerous examples in which cloned genes from one of these organisms has been employed to recover the homologous gene from the alternate organism. Recovery of the FA-A gene can be definitively verified by testing the capacity of the cloned gene to complement the hypersensitivity of FA-A cells to DNA crosslinking agents following transfection. This approach has been chosen because all of the direct mammalian efforts, including an approach recently employed to clone the FA C gene, have thus far failed to recover the FA-A gene. Drosophila was chosen for this study because gene cloning in this organism is facilitated by genetic analysis, the presence of giant polytene chromosomes and a small genome size. It is anticipated that recovery of the human gene will place our collaborators in a position to determine if it can be employed to reverse the lethal effects of Fanconi anemia through gene therapy. This approach is particularly pertinent to Fanconi anemia because the most frequent cause of lethality in this disease is a failure of bone marrow cells to proliferate in late adolescence. This disorder can be reversed in Fanconi children, however, by bone marrow transplantation when a compatible donor is available. If, however, the patients own stem cells could be provided with the normal gene, then each individual could serve as his or her own donor.

The objective of this project is to understand the mechanism by which Drosophila and other multicellular eukaryotes repair covalent interstrand crosslinks between the two strands of the DNA duplex. This type of damage, which can occur as a result of exposure to a number of compounds, has catastrophic implications for the ability of a cell to replicate its genome. It is unique in that crosslinking simultaneously damages sequences on both strands of the duplex, preventing use of either strand as a template for repair.
From genetic screens for mutants uniquely hypersensitive to crosslinking agents, a small group of genes has been identified that appear to be play essential roles in crosslink repair, but to have no essential role in other repair pathways. This phenotype distinguishes these genes from the majority of genes involved in crosslink repair, which are essential components of other major repair pathways. The specific roles in crosslink repair of the four crosslink-specific genes are unknown. The crosslink-sensitive genes, which include mus308, snm1 and two novel mutations, will be studied at the genetic, molecular and biochemical levels to deduce their functional characteristics. The novel mutations will be genetically and molecularly characterized. The snm1 gene, for which there is no mutant allele in Drosophila, will be subjected to targeted and classical mutagenesis until a mutation is obtained. When clones and mutations are available for each of the four genes, the DNA repair phenotypes of all four will be characterized. Epistatic interactions will be determined to position these genes in repair pathways. Interactions at both the genetic and biochemical level will be determined to test the hypothesis that they function as components of a unique aspect of crosslink repair, and biochemical characterization will be performed to understand the molecular role they serve in this process.