The reversible condensation of chromosomes during cell division remains a classic problem in cell biology. Condensation requires the condensin complex 1 in certain experimental systems 2-8, but not in many others 9-15. Anaphase chromosome segregation almost always fails in condensin-depleted cells, leading to the formation of prominent chromatin bridges and cytokinesis failure 4, 9-17. Here, live cell analysis of chicken DT40 cells bearing a conditional knockout of condensin subunit SMC2 reveals that condensin-depleted chromosomes abruptly lose their compact architecture during anaphase and form massive chromatin bridges. The compact chromosome structure can be preserved and anaphase chromosome segregation rescued by preventing the phosphatase targeting subunit RepoMan from recruiting PP1 to chromatin at anaphase onset. This study identifies an activity critical for mitotic chromosome structure that is inactivated by Repo-Man/PP1 during anaphase. This activity, RCA (regulator of chromosome architecture), cooperates with condensin to preserve the characteristic chromosome architecture during mitosis.Mitosis is normal in SMC2 conditional knockout (SMC2 ON/OFF ) chicken DT40 cells grown without doxycycline (SMC2 ON ) 12. By 30 hours after addition of doxycycline to the culture medium (SMC2 OFF ) SMC2 mRNA levels drop at least 160-fold (QRT-PCR, Supplementary Figure 1a) and the protein becomes undetectable in immunoblots. The cells begin to die within 24-48 hours as anaphase chromosome segregation fails and massive chromatin bridges block cytokinesis (Figure 1a-d). The loss of SMC2 is accompanied by loss of other condensin subunits (e.g. CAP-H) from mitotic chromosomes (Supplementary Figure 1b- d).While this anaphase failure is unlikely to be due to defects in cohesin dynamics (see 18 , our unpublished results), it could reflect a loss of DNA topoisomerase II (topo II) function, because topo II localisation is altered in condensin-depleted chromosomes 12,18 , and the activity of extracted Drosophila topo II against an exogenous substrate is decreased following condensin RNAi 18. We therefore examined topo II activity in vivo at a physiological site by quantitating in situ topo II cleavage within the highly characterized 2.1 Mb centromeric α-satellite DXZ1 array of the human X chromosome 19 in four independent SMC2 ON/OFF DT40 hybrid cell lines. No significant differences in topo II activity at this site were found in the presence or absence of condensin (Supplementary Figure 2). Therefore, Correspondence should be addressed to WCE. telephone -44-(0)131-650-7101, fax -44-(0)131-650-7100, Bill.Earnshaw@ed.ac.uk.

Dissection of human centromeres is dif®cult because of the lack of landmarks within highly repeated DNA. We have systematically manipulated a single human X centromere generating a large series of deletion derivatives, which have been examined at four levels: linear DNA structure; the distribution of constitutive centromere proteins; topoisomerase IIa cleavage activity; and mitotic stability. We have determined that the human X major a-satellite locus, DXZ1, is asymmetrically organized with an active subdomain anchored~150 kb in from the Xp-edge. We demonstrate a major site of topoisomerase II cleavage within this domain that can shift if juxtaposed with a telomere, suggesting that this enzyme recognizes an epigenetic determinant within the DXZ1 chromatin. The observation that the only part of the DXZ1 locus shared by all deletion derivatives is a highly restricted region of <50 kb, which coincides with the topoisomerase II cleavage site, together with the high levels of cleavage detected, identify topoisomerase II as a major player in centromere biology.

Summary Topoisomerase II is a major component of mitotic chromosomes and its unique decatenating activity has been implicated in many aspects of chromosome dynamics, of which chromosome segregation is the most seriously affected by loss of topo II activity in living cells. There is considerable evidence that topo II plays a role at the centromere including: the centromere-specific accumulation of topo II protein; cytogenetic/ molecular mapping of topo II catalytic activity to active centromeres; the influence of sumoylated topo II on sister centromere cohesion; and its involvement in the activation of a Mad2-dependent spindle checkpoint. Using a conditional-lethal DNA topoisomerase IIα mutant human cell line we find that topo IIα depletion, while leading to a disorganised metaphase plate, does not have any overt effect on general kinetochore assembly. Fluorescence in situ hybridisation suggested that centromeres segregate normally, most segregation errors being chromatin bridges involving longer chromosome arms. Strikingly, a linear human X centromere-based minichromosome also displayed a significantly increased rate of missegregation. This sensitivity to topo IIα depletion may be linked to structural alterations within the centromere domain, as indicated by a significant shortening of the distance across metaphase sister centromeres, and the abnormal persistence of PICH-coated connections between segregating chromatids.

A genomic probe encompassing most of an esterase gene (E4) that is amplified in insecticideresistant Myzus persicae was hybridized in situ to mitotic and meiotic chromosome preparations of aphid clones of known esterase type and resistance level. Binding, which was detected using the biotin-avidin system located both known types of amplified esterase sequences (E4 and FE4). All except one of the E4-producing clones had a single amplified site, on autosome 3 near the breakpoint of an autosomal 1,3 translocation which previous work had shown to be genetically linked to insecticide resistance. The exceptional clone had two other E4-encoding sites. The most resistant FE4-producing clone (800F) had amplified sequences at five sites (three loci: two homozygous and one heterozygous). Altogether, amplified E4 and/or FE4 sequences were found on four of the five autosome pairs of M. persicae. Possible origins of these multiple loci are discussed.

Karyotypes of permanently parthenogenetic aphids of three species of the genus Trama show great diversity, particularly in the number and distribution of chromosomal elements containing highly repetitive sequences. Sampling at only a few sites in southern England, chromosome number varied from 14 to 23 in T. troglodytes, 9-12 in T. caudata and 10-14 in T. maritima, with some colonies having individuals of more than one karyotype. This variation was paralleled by differences in the number and distribution of rDNA arrays revealed by in situ hybridization. This high intraspecific karyotype diversity contrasts with very low genetic diversity in the same populations, suggesting rapid karyotype evolution. Although T. troglodytes feeds on many species of composite plants there was no evidence of any karyotype-associated host race formation.