X chromosome gene expression is equalized in males and females in a process called dosage compensation. In Drosophila, dosage compensation occurs by increased transcription of X-linked genes in males and is essential for male viability. The product of the maleless (mle) gene is required in males for dosage compensation to occur. The mle protein (Mle) is associated with hundreds of sites on the X chromosome in the male, making it a strong candidate to directly regulate X chromosome transcription. The long term objective of this work is to understand the molecular mechanism of dosage compensation in Drosophila . The experiments proposed focus on the Mle protein. Specific goals are: I. To complete the analysis of mle gene structure and protein expression pattern. II. To define the biochemical activity of Mle protein. III. To characterize X chromosome binding sites of Mle. IV. To determine whether other genes in the dosage compensation genetic hierarchy regulate male-specific activity of Mle. V. To extend these studies toward a more complete understanding of dosage compensation by cloning additional regulatory genes. The amino acid sequence of Mle contains short motifs which place it in a superfamily of ATP binding proteins that are known or putative helicases. The hypothesis that He is a helicase will be tested indirectly, by measuring ATPase activity in the presence of nucleic acids, and directly, by unwinding assays of synthetic duplexes of DNA, RNA and DNA/RNA hybrids. Chromosome binding experiments will map the segments of specific X-linked genes that Mle associates with in vivo. Although Mle is active in males and not in females, Me protein is present in both sexes. The effect of other dosage compensation mutants on Me activity will be tested. X chromosome binding by Me, and any biochemical activities of He that are discovered will be assayed in Sxl, msl-1, msl-2, and mle-3 mutants. An understanding of how dosage compensation occurs may require isolation of the other known regulatory genes and characterization of their products. Isolation of msl-2 will proceed using standard methods for cloning Drosophila genes. It is well documented, especially in Drosophila and humans, that chromosomal balance is necessary for normal development. Mechanisms to equalize X-linked gene expression in males and females exist in many organisms, and disruption of these systems can severely affect development and viability. An understanding of how dosage compensation occurs in Drosophila may have relevance to general mechanisms of chromatin organization and coordinate gene regulation during development.
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