Leishmania mexicana mexicana: Genetic Heterogeneity of Mexican Isolates using Restriction Lenght Polymorphism Analysis of Kinetoplast DNA

We have started to identify and characterise the histone deacetylases of Drosophila melanogaster. Based on their homology with the mammalian enzymes three potential histone deacetylases have been identified. Two of these, dHDACl and dHDAC3 are class I deacetylases (related to yeast Rpd3), whilst the third, dHDAC6 is a class I1 deacetylase (related to yeast Hdal). In addition, we have shown that Drosophila dSIR2 is a novel NAD-dependent histone deacetylase. To facilitate the characterisation of these proteins we have produced stable Schneider cell lines expressing V5-tagged recombinant proteins. dHDACl (520 a.a.) shows an exclusively nuclear distribution, it copurifies with the histone chaperone protein p55, as well as dMBD2/3, and is expressed throughout the whole Drosophila life cycle. We will show that dHDACl regulates its own expression, as the ectopic expression of the recombinant protein shuts off the expression of the endogenous gene. dHDAC3 (438 a.a.) has an apparently fluid cellular distribution, with some cells showing a nuclear distribution, some a cytosolic distribution, and others a combination of the two. Like dHDACl it is expressed throughout the Drosophila life cycle. dHDAC6 (883 a.a., with multiple splice variants) contains two active sites and has an exclusively cytosolic distribution. An antibody based site specificity assay shows all three enzymes are able to deacetylate all lysines of histone H4. The deacetylases in Drosophila are part of distinct protein complexes. dHDACl is found in two complexes (8OOkDa and 200 kDa), whereas dHDAC3 is present in a single 700 kDa complex. Purification of these complexes is underway and preliminary data suggests that these deacetylases associate with several, as yet unidentified, protein partners.