ANALYSIS OF CENTROMERE STRUCTURE IN THE FLY MEGASELIA-SCALARIS (PHORIDAE, DIPTERA) USING CREST SERA, ANTIHISTONE ANTIBODIES, AND A REPETITIVE DNA-PROBE

We have used CREST anti-centromere sera, rabbit anti-histone antibodie s, and repetitive DNA analysis to study centromere structure in the fl y Megaselia scalaris (Phoridae). In a panel of eight CREST sera, four were positive in immunofluorescence experiments for prekinetochores, i e centromeres in interphase nuclei. The access of the antibodies to CR EST antigens may be compromised in the condensed state, since centrome res of prometaphase and metaphase chromosomes remained unstained. When Western blots of embryonic nuclei were probed with these four CREST s era, three of them showed a 17 kDa band. Human CENP-A, likewise recogn ized by the CREST sera, is a 17 kDa protein. The remaining sera were n egative for centromeres although some detected centrosomes and non-his tone chromosomal proteins not confined to the centromeres. The use of antibodies generated against histone H4 acetylated at four different s ites of the N-terminal domain revealed that heterochromatic regions of M scalaris mitotic chromosomes, ie pericentric and NOR-associated seg ments, are hyperacetylated. This is at variance with a variety of othe r systems, where transcriptionally active chromatin is hyperacetylated . Finally, a repetitive 165 base pair fragment was isolated from genom ic DNA of the fly and sequenced. An oligonucleotide from this sequence mapped to the centromere region of interphase nuclei and the pericent ric regions of condensed chromosomes.