A monoclonal antibody against DNA topoisomerase II labels the axial granules of Pleurodeles lampbrush chromosomes

Abstract

By immunizing Balb/c mice with oocyte nuclei of Pleurodeles waltl we obtained a monoclonal antibody, mAb 4A6, that labels distinct globular domains of the lampbrush chromosomal axes of Pleurodeles. These domains are found at corresponding sites of homologous chromosomes, often at telomeric and putative centromeric regions, and appear to be devoid of DNA. Because of these characteristic features it is most likely that the mAb 4A6-positive domains correspond to the central part of the "axial granules" of urodelan lampbrush chromosomes. In immunoblotting analyses mAb 4A6 reacts with a nuclear antigen of approximately Mr 180000 and a structurally nonrelated cytoplasmic protein of Mr 98000, which was not characterized any further. Comparative immunofluorescence and immunoblotting studies with mAb 4A6 and an antiserum against DNA topoisomerase II (topo II) as well as immunodepletion experiments demonstrated that the nuclear 4A6 antigen is topo II. Our results indicate that topo II is not a constituent of a continuous, loop-anchoring scaffold in lampbrush chromosomes of Pleurodeles but, rather, is restricted to the axial granules.

Nucleolar association of pEg7 and XCAP-E, two members ofXenopus laeviscondensin complex in interphase cells

“…Polyclonal anti-topoisomerase II (anti-topoII) antibodies were provided by D. F. Bogenhagen (State University of New York, Stony Brook, USA) (Luke and Bogenhagen, 1989); monoclonal anti-human topoII antibodies were obtained from Calbiochem; monoclonal antiPleurodeles topoII antibodies were a gift from R. Hock (University of Wurzburg) (Hock et al, 1996).…”

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

“…We did not detect cohesins or SYCP3 in the axes of chromosomes condensed into karyospheres in late avian GVs, suggesting that the majority of these cohesion proteins are removed from DNA by condensin action. DNA topo II is absent from the axes of amphibian and avian lampbrush bivalents (Fisher et al 1993, Hock et al 1996, Krasikova et al 2004. All together, these data indicate that the cohesin complex and SC lateral element proteins, as well as the condensins, have a key role in maintaining specific chromosome structure in the lampbrush phase.…”

“…There is, nonetheless, a certain resemblance between PBs and axial granules, which form on amphibian LBC axes (Callan 1986). LBC axial granules in Pleurodeles waltl can fuse with one another and contain DNA topo II (Pyne et al 1995, Hock et al 1996. Centromere and telomere granules on the lampbrush bivalents of Notophthalmus viridescens have neither hnRNP nor snRNP, may reach a diameter of 20 2m, and are proposed to belong to a more Fgeneral class of structures_ (Gall 1992).…”

“…PB size (up to 12 2m diameter) allows analysis of protein distribution and comparative localization by confocal microscopy. Here we performed indirect double immunostaining of PBs on GV spreads using the 4A6 anti-amphibian topo II mAb (Hock et al 1996) and an anti-Rad21 (K854) or anti-SMC3 antibody. These proteins were non-uniformly distributed within PBs, cohesins being localized in the same PB component as topo II (Figure 3a).…”

“…For immunostaining of avian LBCs and ovary sections, we used the following rabbit polyclonal antibodies: K988 anti-human SMC1!, K974 anti-mouse SMC1", K976 anti-human SMC1", K987 antihuman SMC3, K853 and K854 anti-human Rad21, K923 anti-human STAG1, K828 anti-human STAG2, K919 anti-rat SYCP1, K921, K1034 and K1037 antirat SYCP3 (Table 1, Prieto et al 2004), and mouse monoclonal antibody (mAb) 4A6 anti-amphibian topo II (Hock et al 1996). NCBI BLAST was used to search for certain epitopes in the amino acid sequences of chicken proteins or predicted proteins (http://www.ncbi.nlm.nih.gov; see Table 1).…”