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.

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

“…Thus, it is reasonable to expect topo II to be involved in the process of active pol II transcription on the lateral loops of the LBCs. However, immunofluorescence analysis did not reveal direct association of topo II with lateral loops in avian (our data) or in amphibian GVs (Fisher et al 1993;Hock et al 1996). Immunoelectron microscopy studies performed on thin sections of chaffinch GVs showed the presence of topo II in the nucleoplasm.…”

“…In a study of the LBCs of the newt Pleurodeles waltl, Hock et al (1996) demonstrated intense staining of the socalled axial granules with mAb 4A6, which recognizes amphibian topo II. Topoisomerase II was not detectable in other parts of the LBCs (Fisher et al 1993).…”

“…mAb 4A6 against topo II stains the fusing protein bodies but does not stain inactive chromosomes. Bar represents 10 μm cells, which was shown to be topo II (Hock et al 1996). To define the proteins that mAb 4A6 recognizes on avian GV spreads, immunoblotting of chaffinch GV proteins was carried out.…”

“…Immunostaining of avian LBCs and fibroblasts was carried out with the following mouse monoclonal antibodies (mAbs): 4A6 against amphibian topo II (Hock et al 1996), K121 against the trimethylguanosine (TMG) cap of most splicing snRNAs (Oncogene Research Products), Y12 against the Sm core proteins of most splicing snRNPs (Lerner et al 1981), anti-SC35 against the SC35 splicing factor (Fu and Maniatis 1990), H14 (BAbCO) and V22 (kindly donated by U. Scheer) against the phosphorylated C-terminal domain (CTD) of RNA polymerase II, 72B9 and 17C12 against fibrillarin (Pollard et al 1997), No114 against Xenopus Nopp140 and No185 against Xenopus NO38 (Schmidt-Zachmann and Franke 1988) as well as with R288 anti-human coilin rabbit serum (Andrade et al 1993).…”

“…For this reason it has been repeatedly suggested that avian PBs are homologs of these structures, which are known to contain a variety of RNA processing and transcription factors (Gall and Callan 1989;Gall 2000Gall , 2003. Additionally, avian PBs have a morphological similarity with the socalled axial granules, which have been described on LBCs of some amphibians (Callan 1986) and found to contain DNA topoisomerase II (topo II; Hock et al 1996).…”