Analysis of centromere structure in the fly Megaselia scalaris (Phoridae, Diptera) using CREST sera, anti-histone antibodies, and a repetitive DNA probe

We have used CREST anti‐centromere sera, rabbit anti‐histone antibodies, and repetitive DNA analysis to study centromere structure in the fly Megaselia scalaris (Phoridae). In a panel of eight CREST sera, four were positive in immunofluorescence experiments for prekinetochores, ie centromeres in interphase nuclei. The access of the antibodies to CREST antigens may be compromised in the condensed state, since centromeres of prometaphase and metaphase chromosomes remained unstained. When Western blots of embryonic nuclei were probed with these four CREST sera, three of them showed a 17 kDa band. Human CENP‐A, likewise recognized by the CREST sera, is a 17 kDa protein. The remaining sera were negative for centromeres although some detected centrosomes and non‐histone chromosomal proteins not confined to the centromeres. The use of antibodies generated against histone H4 acetylated at four different sites of the N‐terminal domain revealed that heterochromatic regions of M scalaris mitotic chromosomes, ie pericentric and NOR‐associated segments, are hyperacetylated. This is at variance with a variety of other systems, where transcriptionally active chromatin is hyperacetylated. Finally, a repetitive 165 base pair fragment was isolated from genomic DNA of the fly and sequenced. An oligonucleotide from this sequence mapped to the centromere region of interphase nuclei and the pericentric regions of condensed chromosomes.