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

Abstract

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.

Preparation of Insect Chromosomes for Immunolabeling

“…Some studies on insects have involved tissue other than those containing polytene chromosomes, for example, neuroblasts of Drosophila rnelanogaster (Avides andSunkel 1994, Lavender et al 1994. Wffliams andGoldberg 19941, spermatogonia and spermatids of Megasella scalaris (Diptera, Phoridae) [ Wolf et al 1993Wolf et al , 1994b and spermatocytes of crane flies (Bastmeyer et al 1986, LaFountain et al 1992, Ye0 et al 1994. It is of interest to test whether insect chromosomes can be prepared routinely using a cytocen-ge and whether the cytospin preparations can serve as the sub-…”

New Y chromosomes and early stages of sex chromosome differentiation: sex determination in Megaselia

“…But none of the three chromosome pairs reveals itself as a heteromorphic sex chromosome pair under the microscope (figure 1). In mitotic metaphases of M. scalaris, X and Y are indistinguishable when stained with conventional chromosome dyes or with DAPI, with C banding, by comparative genomic hybridisation (CGH) or visualised by scanning electron microscopy: the sex chromosomes are 'homomorphic' Wolf et al 1994;Traut et al 2001). Polytene chromosomes, which usually present much finer cytogenetic details in dipterans, are not sufficiently organized in bands and interbands in this species; they are fuzzy and, hence, inadequate for cytogenetic analysis.…”

The regulation of chromosome segregation via centromere loops

“…(Wolf 1994)). In flies, Drosophila melanogaster (Venkei et al 2012), Megaselia scalaris (Wolf et al 1994), horses (Carbone et al 2006), and human (Salmon et al 1976), sister kinetochores are also separated by 1 micron. Even in the smallest eukaryote, Ostreococcus tauri, sister kinetochore separation is 400 nm (Gan et al 2011) (Table 1).…”