Jane E. Selegue

The Regulation and Evolution of a Genetic Switch Controlling Sexually Dimorphic Traits in Drosophila

Sexually dimorphic traits play key roles in animal evolution and behavior. Little is known, however, about the mechanisms governing their development and evolution. One recently evolved dimorphic trait is the male-specific abdominal pigmentation of Drosophila melanogaster, which is repressed in females by the Bric-à-brac (Bab) proteins. To understand the regulation and origin of this trait, we have identified and traced the evolution of the genetic switch controlling dimorphic bab expression. We show that the HOX protein Abdominal-B (ABD-B) and the sex-specific isoforms of Doublesex (DSX) directly regulate a bab cis-regulatory element (CRE). In females, ABD-B and DSX(F) activate bab expression whereas in males DSX(M) directly represses bab, which allows for pigmentation. A new domain of dimorphic bab expression evolved through multiple fine-scale changes within this CRE, whose ancestral role was to regulate other dimorphic features. These findings reveal how new dimorphic characters can emerge from genetic networks regulating pre-existing dimorphic traits.

Ectopic gene expression and homeotic transformations in arthropods using recombinant Sindbis viruses

BACKGROUND The morphological diversity of arthropods makes them attractive subjects for studying the evolution of developmental mechanisms. Comparative analyses suggest that arthropod diversity has arisen largely as a result of changes in expression patterns of genes that control development. Direct analysis of how a particular gene functions in a given species during development is hindered by the lack of broadly applicable techniques for manipulating gene expression. RESULTS We report that the Arbovirus Sindbis can be used to deliver high levels of gene expression in vivo in a number of non-host arthropod species without causing cytopathic effects in infected cells or impairing development. Using recombinant Sindbis virus, we investigated the function of the homeotic gene Ultrabithorax in the development of butterfly wings and beetle embryos. Ectopic Ultrabithorax expression in butterfly forewing imaginal discs was sufficient to cause the transformation of characteristic forewing properties in the adult, including scale morphology and pigmentation, to those of the hindwing. Expression of Ultrabithorax in beetle embryos outside of its endogenous expression domain affected normal development of the body wall cuticle and appendages. CONCLUSIONS The homeotic genes have long been thought to play an important role in the diversification of arthropod appendages. Using recombinant Sindbis virus, we were able to investigate homeotic gene function in non-model arthropod species. We found that Ultrabithorax is sufficient to confer hindwing identity in butterflies and alter normal development of anterior structures in beetles. Recombinant Sindbis virus has broad potential as a tool for analyzing how the function of developmental genes has changed during the diversification of arthropods.

Recombinant 70-kDa protein from the amino-terminal region of rat fibronectin inhibits binding of fibronectin to cells and bacteria.

Binding of fibronectin to substrate-attached cells and to Staphylococcus aureus is mediated by the amino-terminal 70-kDa portion of fibronectin. The 70-kDa amino-terminus is composed of nine type I and two type II internal homology units, each containing two intrachain disulfide bonds. The exact structural features of the 70-kDa amino-terminus that are necessary for binding to cells and bacteria are not known. We characterized a recombinant 70-kDa protein from the amino-terminus of rat fibronectin using a baculovirus expression system. Recombinant 70-kDa (r70kDa) protein was easily purified in high amounts from the conditioned medium by affinity chromatography on gelatin-agarose. Secretion was much less when N-linked glycosylation was blocked by tunicamycin. Like the native fragment, the r70kDa protein contains intrachain disulfide bonds. In addition, the r70kDa protein was indistinguishable from the nonrecombinant 70-kDa fragment in its ability to compete for binding sites on fibroblasts and S. aureus. Thus, the r70kDa protein retains the important functional characteristics of the native fragment. This expression system is well adapted to studying the structural features important for the interaction of 70-kDa protein with cells.

ESTABLISHMENT AND CHARACTERIZATION OF ADENOSINE DEAMINASE (ADA)-DEFICIENT T CELL LINES

Congenital deficiency of the purine metabolic enzyme ADA causes severe combined immunodeficiency (SCID). The profound T lymphopenia characteristic of this disease has limited direct investigation of the cell most affected by deficiency of ADA. We have been successful in establishing both IL-2-dependent, non-transformed and HTLV-1 transformed T cell lines from eight consecutive ADA-deficient SCID patients. The lines obtained by either method are mature T cells by surface phenotype. These lines all display hypersensitivity to deoxyadenosine, characteristic of ADA deficiency, which is not reversed by deoxycytidine. Clones obtained by limiting dilution of one line show multiple unique rearrangeents of the T cell receptor beta gene suggesting clonal diversity among the circulating T cells of this patient, despite lymphopenia and SCID. Lines from 5 patients, all under three years old, have less than 1% normal ADA activity. Pharmacologic inhibitor studies suggest that this remaining activity is attributable to a non-specific aminohydrolase. In contrast, cells from two children ages 11 and 13 years are partially deficient with up to 10% normal ADA activity. An eighth SCID patient, previously found to totally lack erythrocyte ADA has approximately 50% normal ADA in a line derived from thymocytes. Analysis of the molecular basis for ADA deficiency in these lines shows that the majority have a grossly intact ADA gene, contain normal size ADA mRNA (1.6kb) and produce an ADA protein that is catalytically defective.