Localization of RAP1 and topoisomerase II in nuclei and meiotic chromosomes of yeast

Abstract

Abstract. Topoisomerase II (topolD and RAP1 (Repressor Activator Protein 1) are two abundant nuclear proteins with proposed structural roles in the higherorder organization of chromosomes. Both proteins cofractionate as components of nuclear scaffolds from vegetatively growing yeast cells, and both proteins are present as components of pachytene chromosomes, cofractionating with an insoluble subfraction of meiotic nuclei. Immunolocalization using antibodies specific for topolI shows staining of an axial core of the yeast meiotic chromosome, extending the length of the synaptonemal complex. RAP1, on the other hand, is located at the ends of the paired bivalent chromosomes, consistent with its ability to bind telomeric sequences in vitro. In interphase nuclei, again in contrast to antitopolI, anti-RAP1 gives a distinctly punctate staining that is located primarily at the nuclear periphery. Approximately 16 brightly staining foci can be identified in a diploid nucleus stained with anti-RAP1 antibodies, suggesting that telomeres are grouped together, perhaps through interaction with the nuclear envelope.

Ectopic expression of inactive forms of yeast DNA topoisomerase II confers resistance to the anti-tumour drug, etoposide

“…Because our antibodies cannot distinguish between truncated and wild-type topoisomerase II forms, we always detect low-level immunofluorescence of the endogenous, exclusively nuclear wild-type enzyme (see Figure 7a; Klein et al, 1992).…”

High copy number suppression of the meiotic arrest caused by a dmc1 mutation: REC114 imposes an early recombination block and RAD54 promotes a DMC1‐independent DSB repair pathway

“…Spread meiotic nucleoids were prepared as described by Klein et al (1992) except H 2 O was used in place of 1% Lipsol detergent and formaldehyde was used at 2% rather than at 4%. These modi®cations result in somewhat inferior preparations compared to the standard method, but were necessary for examination of tubulin staining patterns because microtubules are sensitive to Lipsol treatment.…”

Meiotic behaviours of chromosomes and microtubules in budding yeast: relocalization of centromeres and telomeres during meiotic prophase

“…To achieve this goal, we have determined the nuclear locations of telomeres and centromeres in synchronous populations of meiotic cells. Telomeres were detected using the chromosomal integration of GFP-Rap1 fusion construct (see Experimental procedures) or an antibody against Rap1 protein (Klein et al 1992), which has been shown to co-localize with telomeric repeats by combined fluorescence in situ hybridization and immunofluorescence studies (Gotta et al 1996). The localization of Rap1 protein at the chromosome ends can be seen in spread preparations of pachytene chromosomes (Fig.…”