Molecular and biochemical analysis of the {\it Drosophila\/} segmentation gene {\it runt\/}

Genetic evidence has indicated that the segmentation gene runt plays a key role in regulating gene expression of the pair-rule genes hairy, even-skipped, and fushi tarazu. In contrast to other pair-rule genes, sequence data of the runt open reading frame did not reveal homologies to DNA-binding motifs of known transcriptional regulatory proteins. This thesis project examined several properties of the runt gene based on the sequence of the transcription unit, including the subcellular localization of the protein in vivo, its ability to bind DNA, and the functionality of a putative nucleotide binding domain.^ A runt-specific antibody was generated and used to demonstrate that runt is localized in the nucleus. Since the precise overlap of the pair-rule stripes is thought to be critical for the determination of cellular identity along the anterior-posterior axis, phasing of early runt expression in the blastoderm was examined with regard to the segmentation genes hairy, even-skipped, and fushi tarazu. runt was also expressed at later stages of embryogenesis, including expression in neuroblasts, and ganglion mother cells of the developing nervous system. Expression at this stage was required for the subsequent formation of specific neurons and runt was extensively expressed in the central and peripheral nervous systems.^ Several experiments were done to address the biochemical function of the runt protein. A direct interaction of runt with DNA was first examined. Although bacterial expressed runt was found to bind dsDNA-cellulose, subsequent experiments failed to detect sequence-specific interactions with DNA. Inter-species conservation of the putative nucleotide binding domain suggested that this region was functionally important, and runt protein bound a labeled ATP analog with high affinity in vitro. Finally, the effect of substitution of a critical residue of the nucleotide binding domain on runt activity was examined in vivo. Ectopic expression of the mutant protein indicated that this conserved substitution altered, but did not eliminate, runt activity as evaluated by segmentation phenotype and viability. ^

A genetic characterization of {\it runt\/} activity during {\it Drosophila\/} embryogenesis

The Drosophila melanogaster gene runt encodes a novel transcriptional regulator that was originally identified on the basis of its key role in embryonic pattern formation. For my thesis I undertook a genetic analysis of runt activity to identify loci that interact with this unique transcriptional regulator. Specifically, I screened the genome with deficiencies for loci that interact with runt in a dose-dependent fashion during early embryogenesis. From this screen I discovered a vital dose-dependent interaction between runt and the achaete-scute complex (AS-C). The characterization of this interaction led to the exciting discovery of important roles for runt in sex determination and neurogenesis (Duffy and Gergen 1991, Duffy et al. 1991). I demonstrated that in sex determination runt is necessary for the normal transcriptional activation of the master sex-determining gene Sx1 and has all the properties of an X:A numerator element. I also showed that runt is required during the early stages of neurogenesis for the normal development of a subset of CNS ganglion mother cells and neurons. In addition, the screen, which focused on the identification and characterization of maternal loci that influence the activity of runt during segmentation, identified several new maternal loci, one of which affects the activity of the maternal posterior group genes on embryonic pattern formation. ^