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Development of a reporter gene assay to identify control elements required for dosage compensation in Drosophila Melanogaster : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University
Dosage compensation (equalisation of X-linked gene products) occurs in Drosophila melanogaster by a two-fold transcriptional increase of X-linked gene expression in the male. This involves the binding of four proteins, MSL-1, MSL-2, MSL-3 and MLE (collectively known as the MSLs), to hundreds of sites along the length of the male X. The MSLs are thought to recruit MOF, a histone acetyl transferase, which facilitates the increase in transcriptional activity of X-linked genes. The DNA sequences required to target the MSL complex to the X chromosome (known as dosage compensation regulatory elements, or DCREs) remain elusive, despite numerous attempts over the last ten years to identify them. DCREs are thought to be present at multiple sites along the length of the X chromosome, as antibodies to the MSLs bind to hundreds of sites along the X, and autosomal genes transduced to the X usually become dosage compensated. The first objective of this study was to develop a reporter gene assay to screen for DCREs that would minimise problems previously encountered. A construct consisting of the constitutive armadillo promoter fused to the lacZ reporter gene (called arm-lacZ) was flanked by insulator elements which block the repressive effects of the autosomal chromatin environment. Fragments of X-linked DNA were inserted upstream of the armadillo promoter with the premise that males carrying one copy of an autosomal insertion of this construct would express twice the level of ß-galactosidase as females. Transgenic flies carrying autosomal insertions of X-linked fragments plus arm-lacZ were generated and one dose males and females were assayed for ß-galactosidase activity using a spectrophotometric assay. In all cases, males and females expressed the same level of lacZ. This suggests that no DCREs that could confer dosage compensation onto arm-lacZ were present in the X-linked fragments. arm-lacZ is capable of being dosage compensated as males and females carrying one copy of an X-linked insertion of arm-lacZ produce a 2:1 male to female ratio. This implies that DCREs of the 'strength' required to dosage compensate arm-lacZ are rarer than previously thought. A second method of dosage compensation that is independent of the MSLs is thought to occur in Drosophila. The X-linked gene runt is dosage compensated in the absence of the MSLs. It is possible that runt is sex specifically regulated by the female specific Sex lethal protein (Sxl). Sxl down-regulates msl-2 in females by binding to (U)8 or A(U)7 sequences in the msl-2 5' and 3' untranslated regions (UTRs) of the mRNA. runt mRNA contains three Sxl binding sites in its 3' UTR, as do 20 other X-linked genes. The second objective of this project was to determine if Sxl could down regulate a gene in females, purely by the addition of three Sxl binding sites to the 3'UTR. Sxl binding sites were inserted into the 3'UTR of arm-lacZ in the form of a 40 bp synthetic linker containing three of the sites, and also as a 170 bp fragment from the runt 3' UTR. ß-galactosidase assays of flies carrying the Sxl binding sites from runt showed that males expressed an average of 1.31 to 1.46 times the level of lacZ than females. This shows that Sxl can down-regulate a gene if there are Sxl binding sites in its 3' UTR, however, to achieve two-fold regulation, additional factors may be required, or topologically, the sites may not have been in the right position in the 3' UTR for optimal activity of Sxl. Flies carrying the synthetic linker expressed the same level of ß-galactosidase in both sexes which suggests that either additional elements within the 3' UTR are required, or that the spacing between the sites is critical for the action of Sxl.