Centromeric protein bodies on avian lampbrush chromosomes contain a protein detectable with an antibody against DNA topoisomerase II

Abstract

In the oocyte nuclei (germinal vesicle or GV) of a variety of avian species, prominent spherical entities termed protein bodies (PBs) arise at the centromeric regions of the lampbrush chromosomes (LBCs). In spite of the obvious protein nature of PBs, nothing is known about their composition. We show that an antibody against DNA topoisomerase II (topo II), the DNA unwinding enzyme, recognizes PBs from chaffinch and pigeon oocytes. In later chaffinch oocytes, the PBs fuse to form a karyosphere, which is also labeled by the anti-topo II antibody. Furthermore, we show that proteins characteristic of Cajal bodies and B-snurposomes are not found in PBs, despite morphological similarities among these structures. Using immunoelectron microscopy and immunofluorescent laser scanning microscopy we demonstrated that topo II localizes predominantly in the dense material of PBs. Two antigens of approximately 170 kDa (which corresponds to topo II) and approximately 100 kDa were revealed with the antibody against topo II on immunoblots of avian GV proteins. We propose that the smaller protein results from oocyte specific topo II cleavage, since it was not detected in nuclei from testis cells. This represents the first report of a defined protein in the centromeric PBs on avian LBCs.

Cohesion proteins are present in centromere protein bodies associated with avian lampbrush chromosomes

“…These structures correspond to PBs, as determined from phase-contrast images (not shown). Similar staining was observed for 4A6 anti-topo II mAb on frozen sections (Figure 3b 00 ), concurring with immunogold labelling of avian GV thin sections (Krasikova et al 2004). …”

“…In chaffinch, pigeon and duck oocytes, we recently demonstrated that DNA topoisomerase II (topo II) is located predominantly in the dense component of PBs but not in the vacuoles, as shown by immunoelectron microscopy (Krasikova et al 2004). PB size (up to 12 2m diameter) allows analysis of protein distribution and comparative localization by confocal microscopy.…”

“…In pigeon and chaffinch GVs, PBs form adjacent to the chromatin blocks of centromeric satellite repeats PR1 and FCP, respectively (Solovei et al 1996, Saifitdinova et al 2003. The PBs do not contain splicing factors, splicing RNA or transcribing RNA polymerase II (Saifitdinova et al 2003, Krasikova et al 2004, and thus differ from Cajal bodies and B-snurposomes (or speckles). The role of the PBs remains unclear, but it seems that these structures reflect a thus-far unknown centromere function.…”

“…The only PB component reported to date is DNA topoisomerase II (topo II; Krasikova et al 2004), which is known to be involved in topological organization of chromatin (Earnshaw et al 1985). We thus hypothesized that PBs may contain other proteins implicated in structural maintenance of chromosomes and important for centromere function.…”

The karyosphere capsule inRana temporariaoocytes contains structural and DNA-binding proteins

“…In meiotic cells, in particular, in diplotene oocytes of mice, topo II is abundant in the nucleoplasm, but absent in the central body of the mouse karyosphere [30]. In birds, topo II is concentrated in protein bodies associated with the centromere regions of lampbrush chromosomes and, in the late diplotene, in the central body of the bird karyosphere [54]. Unlike the mouse, the central body of the karyosphere of birds arises due to the fusion of these specific centromere protein bodies following the lampbrush stage.…”