Extracentromeric connections between sister chromatids demonstrated in human chromosomes induced to condense asymmetrically

Summary Extracentromeric chromatin fibers were proposed to hold sister chromatids together in mitotic chromosomes examined by electron microscopy, but their existence in living cells has not been demonstrated yet. We have performed an in vitro BrdU-H33258 treatment which induced a differential rate of condensation to each sister chromatid, thus producing asymmetrically condensing chromosomes. The fast condensing chromatid pulled the slower sister one, both bending in parallel. Bent chromatids appeared reciprocally connected by loops of chromatin fibers, suggesting they were the links which permitted the physical interplay between the differently condensing chromatids. When sister chromatid exchanges (SCE) intercalated a fast-condensing fragment in the slow-condensing chromatid or vice versa, the chromosome inverted its curvature at the SCE-point.     

Human Wapl is a cohesin-binding protein that promotes sister-chromatid resolution in mitotic prophase

BACKGROUND: The linkage between duplicated chromosomes (sister chromatids) is established during S phase by the action of cohesin, a multisubunit complex conserved from yeast to humans. Most cohesin dissociates from chromosome arms when the cell enters mitotic prophase, leading to the formation of metaphase chromosomes with two cytologically discernible chromatids. This process is known as sister-chromatid resolution. Although two mitotic kinases have been implicated in this process, it remains unknown exactly how the cohesin-mediated linkage is destabilized at a mechanistic level. RESULTS: The wings apart-like (Wapl) protein was originally identified as a gene product that potentially regulates heterochromatin organization in Drosophila melanogaster. We show that the human ortholog of Wapl is a cohesin-binding protein that facilitates cohesin's timely release from chromosome arms during prophase. Depletion of Wapl from HeLa cells causes transient accumulation of prometaphase-like cells with chromosomes that display poorly resolved sister chromatids with a high level of cohesin. Reduction of cohesin relieves the Wapl-depletion phenotype, and depletion of Wapl rescues premature sister separation observed in Sgo1-depleted or Esco2-depleted cells. Conversely, overexpression of Wapl causes premature separation of sister chromatids. Wapl physically associates with cohesin in HeLa-cell nuclear extracts. Remarkably, in vitro reconstitution experiments demonstrate that Wapl forms a stoichiometric, ternary complex with two regulatory subunits of cohesin, implicating its noncatalytic function in inactivating cohesin's ability to interact with chromatin. CONCLUSIONS: Wapl is a new regulator of sister chromatid resolution and promotes release of cohesin from chromosomes by directly interacting with its regulatory subunits.     

Sister Chromatid Cohesion

During S phase, not only does DNA have to be replicated, but also newly synthesized DNA molecules have to be connected with each other. This sister chromatid cohesion is essential for the biorientation of chromosomes on the mitotic or meiotic spindle, and is thus an essential prerequisite for chromosome segregation. Cohesion is mediated by cohesin complexes that are thought to embrace sister chromatids as large rings. Cohesin binds to DNA dynamically before DNA replication and is converted into a stably DNA-bound form during replication. This conversion requires acetylation of cohesin, which in vertebrates leads to recruitment of sororin. Sororin antagonizes Wapl, a protein that is able to release cohesin from DNA, presumably by opening the cohesin ring. Inhibition of Wapl by sororin therefore “locks” cohesin rings on DNA and allows them to maintain cohesion for long periods of time in mammalian oocytes, possibly for months or even years.DNA replication during the synthesis (S) phase generates identical DNA molecules, which, in their chromatinized form, are called sister chromatids. The pairs of sister chromatids remain united as part of one chromosome during the subsequent gap (G₂) phase and during early mitosis, in prophase, prometaphase, and metaphase. During these stages of mitosis chromosomes condense, in most eukaryotes the nuclear envelope breaks down, and in all species chromosomes are ultimately attached to both poles of the mitotic spindle. Only once this biorientation has been achieved for all chromosomes, the sister chromatids are separated from each other in anaphase and transported toward opposite spindle poles of the mother cell, enabling its subsequent division into two genetically identical daughter cells.This series of events critically depends on the fact that sister chromatids remain physically connected with each other from S phase until metaphase. This physical connection, called sister chromatid cohesion, opposes the pulling forces that are generated by microtubules that attach to kinetochores and thereby enables the biorientation of chromosomes on the mitotic spindle (Tanaka et al. 2000b). Without cohesion, sister chromatids could therefore not be segregated symmetrically between the forming daughter cells, resulting in aneuploidy. For the same reasons, cohesion is essential for chromosome segregation in meiosis I and meiosis II. Cohesion defects in human oocytes can lead to aneuploidy, which is thought to be the major cause of spontaneous abortion, because only a few types of aneuploidy are compatible with viability, such as trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome), and trisomy 13 (Patau syndrome) (Hunt and Hassold 2010). Studying the mechanisms of cohesion is therefore essential for understanding how the genome is passed properly from one cell generation to the next.In addition, sister chromatid cohesion facilitates the repair of DNA double-strand breaks in cells that have replicated their DNA, where such breaks can be repaired by a homologous recombination mechanism that uses the undamaged sister chromatid as a template (for review, see Watrin et al. 2006). Furthermore, mutations in the proteins that are required for sister chromatid cohesion can cause defects in chromatin structure and gene regulation, and can in rare cases lead to congenital developmental disorders, called Cornelia de Lange syndrome, Roberts/SC Phocomelia syndrome, and Warsaw Breakage syndrome (for review, see Mannini et al. 2010).     

Chromosome behavior after laser microirradiation of a single kinetochore in mitotic PtK2 cells

The role of the kinetochore in chromosome movement was studied by 532- nm wavelength laser microirradiation of mitotic PtK2 cells. When the kinetochore of a single chromatid is irradiated at mitotic prometaphase or metaphase, the whole chromosome moves towards the pole to which the unirradiated kinetochore is oriented, while the remaining chromosomes congregate on the metaphase plate. The chromatids of the irradiated chromosome remain attached to one another until anaphase, at which time they separate by a distance of 1 or 2 micrometers and remain parallel to each other, not undergoing any poleward separation. Electron microscopy shows that irradiated chromatids exhibit either no recognizable kinetochore structure or a typical inactive kinetochore in which the tri-layer structure is present but has no microtubules associated with it. Graphical analysis of the movement of the irradiated chromosome shows that the chromosome moves to the pole rapidly with a velocity of approximately 3 micrometers/min. If the chromosome is close to one pole at irradiation, and the kinetochore oriented towards that pole is irradiated, the chromosome moves across the spindle to the opposite pole. The chromosome is slowed down as it traverses the equatorial region, but the velocity in both half-spindles is approximately the same as the anaphase velocity of a single chromatid. Thus a single kinetochore moves twice the normal mass of chromatin (two chromatids) at the same velocity with which it moves a single chromatid, showing that the velocity with which a kinetochore moves is independent, within limits, of the mass associated with it.     

Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast

In eukaryotic cells, replicated DNA strands remain physically connected until their segregation to opposite poles of the cell during anaphase. This "sister chromatid cohesion" is essential for the alignment of chromosomes on the mitotic spindle during metaphase. Cohesion depends on the multisubunit cohesin complex, which possibly forms the physical bridges connecting sisters. Proteolytic cleavage of cohesin's Sccl subunit at the metaphase to anaphase transition is essential for sister chromatid separation and depends on a conserved protein called separin. We show here that separin is a cysteine protease related to caspases that alone can cleave Sccl in vitro. Cleavage of Sccl in metaphase arrested cells is sufficient to trigger the separation of sister chromatids and their segregation to opposite cell poles.     

Involvement of chromatid cohesiveness at the centromere and chromosome arms in meiotic chromosome segregation: A cytological approach

Kinetochores and chromatid cores of meiotic chromosomes of the grasshopper species Arcyptera fusca and Eyprepocnemis plorans were differentially silver stained to analyse the possible involvement of both structures in chromatid cohesiveness and meiotic chromosome segregation. Special attention was paid to the behaviour of these structures in the univalent sex chromosome, and in B univalents with different orientations during the first meiotic division. It was observed that while sister chromatid of univalents are associated at metaphase I, chromatid cores are individualised independently of their orientation. We think that cohesive proteins on the inner surface of sister chromatids, and not the chromatid cores, are involved in the chromatid cohesiveness that maintains associated sister chromatids of bivalents and univalents until anaphase I. At anaphase I sister chromatids of amphitelically oriented B univalents or spontaneous autosomal univalents separate but do not reach the poles because they remain connected at the centromere by a long strand which can be visualized by silver staining, that joins stretched sister kinetochores. This strand is normally observed between sister kinetochores of half-bivalents at metaphase II and early anaphase II. We suggest that certain centromere proteins that form the silver-stainable strand assure chromosome integrity until metaphase II. These cohesive centromere proteins would be released or modified during anaphase II to allow normal chromatid segregation. Failure of this process during the first meiotic division could lead to the lagging of amphitelically oriented univalents. Based on our results we propose a model of meiotic chromosome segregation. During mitosis the cohesive proteins located at the centromere and chromosome arms are released during the same cellular division. During meiosis those proteins must be sequentially inactivated, i.e. those situated on the inner surface of the chromatids must be eliminated during the first meiotic division while those located at the centromere must be released during the second meiotic division.by D.P. Bazett-Jones     

Genes occupy a fixed and symmetrical position on sister chromatids

A high-resolution fluorescence methodology for nonisotopic in situ hybridization was applied to determine the positions occupied by several single-copy genes, DNA sequences, and integrated viral genomes on sister chromatids. The lateral and longitudinal mapping of the probes was performed on prometaphase and metaphase chromosomes. A fixed lateral position, exterior or median in relation to the longitudinal axis of the chromatids, was observed for a given probe, with a symmetrical position of the double fluorescent spots. This position appears to be independent of chromosome condensation stage from prometaphase to metaphase. These observations suggest an opposite helical-handedness conformation of DNA on both chromatids with a mirror symmetry. They support the model of chromosome packaging recently proposed by Boy de la Tour and Laemmli. Moreover, our results indicate that the last stages of chromosome condensation occur by packing down the coils without further coiling.     

Visualization of early chromosome condensation: a hierarchical folding, axial glue model of chromosome structure

Current models of mitotic chromosome structure are based largely on the examination of maximally condensed metaphase chromosomes. Here, we test these models by correlating the distribution of two scaffold components with the appearance of prophase chromosome folding intermediates. We confirm an axial distribution of topoisomerase IIalpha and the condensin subunit, structural maintenance of chromosomes 2 (SMC2), in unextracted metaphase chromosomes, with SMC2 localizing to a 150-200-nm-diameter central core. In contrast to predictions of radial loop/scaffold models, this axial distribution does not appear until late prophase, after formation of uniformly condensed middle prophase chromosomes. Instead, SMC2 associates throughout early and middle prophase chromatids, frequently forming foci over the chromosome exterior. Early prophase condensation occurs through folding of large-scale chromatin fibers into condensed masses. These resolve into linear, 200-300-nm-diameter middle prophase chromatids that double in diameter by late prophase. We propose a unified model of chromosome structure in which hierarchical levels of chromatin folding are stabilized late in mitosis by an axial "glue."     

Mouse satellite DNA, centromere structure, and sister chromatid pairing

The experiments described were directed toward understanding relationships between mouse satellite DNA, sister chromatid pairing, and centromere function. Electron microscopy of a large mouse L929 marker chromosome shows that each of its multiple constrictions is coincident with a site of sister chromatid contact and the presence of mouse satellite DNA. However, only one of these sites, the central one, possesses kinetochores. This observation suggests either that satellite DNA alone is not sufficient for kinetochore formation or that when one kinetochore forms, other potential sites are suppressed. In the second set of experiments, we show that highly extended chromosomes from Hoechst 33258-treated cells (Hilwig, I., and A. Gropp, 1973, Exp. Cell Res., 81:474-477) lack kinetochores. Kinetochores are not seen in Miller spreads of these chromosomes, and at least one kinetochore antigen is not associated with these chromosomes when they were subjected to immunofluorescent analysis using anti-kinetochore scleroderma serum. These data suggest that kinetochore formation at centromeric heterochromatin may require a higher order chromatin structure which is altered by Hoechst binding. Finally, when metaphase chromosomes are subjected to digestion by restriction enzymes that degrade the bulk of mouse satellite DNA, contact between sister chromatids appears to be disrupted. Electron microscopy of digested chromosomes shows that there is a significant loss of heterochromatin between the sister chromatids at paired sites. In addition, fluorescence microscopy using anti-kinetochore serum reveals a greater inter-kinetochore distance than in controls or chromosomes digested with enzymes that spare satellite. We conclude that the presence of mouse satellite DNA in these regions is necessary for maintenance of contact between the sister chromatids of mouse mitotic chromosomes.     

Immunocytology of chiasmata and chromosomal disjunction at mouse meiosis

Immunocytological and in situ hybridization evidence supports the hypothesis that at meiosis of chiasmate organisms, chromosomal disjunction and reductional segregation of sister centromeres are integrated with synaptonemal complex functions. The Mr 125,000 synaptic protein, Syn1, present between cores of paired homologous chromosomes during pachytene of meiotic prophase, is lost from synaptonemal complexes coordinately with homolog separation at diplotene. Separation is constrained by exchanges between non-sister chromatids, the chiasmata. We show that the Mr 30,000 chromosomal core protein, Cor1, associated with sister chromatid pairs, remains an axial component of post-pachytene chromosomes until metaphase I. We demonstrate that at this time the chromatin loops are still attached to their cores. A reciprocal exchange event between two homologous non-sister chromatids is therefore immobilized by anchorage of sister chromatids to their respective cores. Cores thus contribute to the sister chromatid cohesiveness required for maintenance of chiasmata and proper chromosomal disjunction. Cor1 protein accumulates in juxtaposition to pairs of sister centromeres during metaphase I. Presumably, independent movement of sister centromeres at anaphase I is restricted by Cor1 anchorage. That reductional separation of sister centromeres is mediated by Cor1, is supported by the dissociation of Cor1 from separating sister centromeres at anaphase II and by its absence from mitotic anaphases.     

A role for Drosophila SMC4 in the resolution of sister chromatids in mitosis

BACKGROUND: Faithful segregation of the genome during mitosis requires interphase chromatin to be condensed into well-defined chromosomes. Chromosome condensation involves a multiprotein complex known as condensin that associates with chromatin early in prophase. Until now, genetic analysis of SMC subunits of the condensin complex in higher eukaryotic cells has not been performed, and consequently the detailed contribution of different subunits to the formation of mitotic chromosome morphology is poorly understood. RESULTS: We show that the SMC4 subunit of condensin is encoded by the essential gluon locus in Drosophila. DmSMC4 contains all the conserved domains present in other members of the structural-maintenance-of-chromosomes protein family. DmSMC4 is both nuclear and cytoplasmic during interphase, concentrates on chromatin during prophase, and localizes to the axial chromosome core at metaphase and anaphase. During decondensation in telophase, most of the DmSMC4 leaves the chromosomes. An examination of gluon mutations indicates that SMC4 is required for chromosome condensation and segregation during different developmental stages. A detailed analysis of mitotic chromosome structure in mutant cells indicates that although the longitudinal axis can be shortened normally, sister chromatid resolution is strikingly disrupted. This phenotype then leads to severe chromosome segregation defects, chromosome breakage, and apoptosis. CONCLUSIONS: Our results demonstrate that SMC4 is critically important for the resolution of sister chromatids during mitosis prior to anaphase onset.     

Sororin, a substrate of the anaphase-promoting complex, is required for sister chromatid cohesion in vertebrates

We have identified a regulator of sister chromatid cohesion in a screen for cell cycle-controlled proteins. This 35 kDa protein is degraded through anaphase-promoting complex (APC)-dependent ubiquitination in G1. The protein is nuclear in interphase cells, dispersed from the chromatin in mitosis, and interacts with the cohesin complex. In Xenopus embryos, overexpression of the protein causes failure to resolve and segregate sister chromatids in mitosis and an increase in the level of cohesin associated with metaphase chromosomes. In cultured cells, depletion of the protein causes mitotic arrest and complete failure of sister chromatid cohesion. This protein is thus an essential, cell cycle-dependent mediator of sister chromatid cohesion. Based on sequence analysis, this protein has no apparent orthologs outside of the vertebrates. We speculate that the protein, which we have named sororin, regulates the ability of the cohesin complex to mediate sister chromatid cohesion, perhaps by altering the nature of the interaction of cohesin with the chromosomes.     

Sister chromatid resolution: a cohesin releasing network and beyond

When chromosomes start to assemble in mitotic prophase, duplicated chromatids are not discernible within each chromosome. As condensation proceeds, they gradually show up, culminating in two rod-shaped structures apposed along their entire length within a metaphase chromosome. This process, known as sister chromatid resolution, is thought to be a prerequisite for rapid and synchronous separation of sister chromatids in anaphase. From a mechanistic point of view, the resolution process can be dissected into three distinct steps: (1) release of cohesin from chromosome arms; (2) formation of chromatid axes mediated by condensins; and (3) untanglement of inter-sister catenation catalyzed by topoisomerase II (topo II). In this review article, we summarize recent progress in our understanding the molecular mechanisms of sister chromatid resolution with a major focus on its first step, cohesin release. An emerging idea is that this seemingly simple step is regulated by an intricate network of positive and negative factors, including cohesin-binding proteins and mitotic kinases. Interestingly, some key factors responsible for cohesin release in early mitosis also play important roles in controlling cohesin functions during interphase. Finally, we discuss how the step of cohesin release might mechanistically be coordinated with the actions of condensins and topo II.     

Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells

Cohesion between sister chromatids is essential for their bi-orientation on mitotic spindles. It is mediated by a multisubunit complex called cohesin. In yeast, proteolytic cleavage of cohesin's alpha kleisin subunit at the onset of anaphase removes cohesin from both centromeres and chromosome arms and thus triggers sister chromatid separation. In animal cells, most cohesin is removed from chromosome arms during prophase via a separase-independent pathway involving phosphorylation of its Scc3-SA1/2 subunits. Cohesin at centromeres is refractory to this process and persists until metaphase, whereupon its alpha kleisin subunit is cleaved by separase, which is thought to trigger anaphase. What protects centromeric cohesin from the prophase pathway? Potential candidates are proteins, known as shugoshins, that are homologous to Drosophila MEI-S332 and yeast Sgo1 proteins, which prevent removal of meiotic cohesin complexes from centromeres at the first meiotic division. A vertebrate shugoshin-like protein associates with centromeres during prophase and disappears at the onset of anaphase. Its depletion by RNA interference causes HeLa cells to arrest in mitosis. Most chromosomes bi-orient on a metaphase plate, but precocious loss of centromeric cohesin from chromosomes is accompanied by loss of all sister chromatid cohesion, the departure of individual chromatids from the metaphase plate, and a permanent cell cycle arrest, presumably due to activation of the spindle checkpoint. Remarkably, expression of a version of Scc3-SA2 whose mitotic phosphorylation sites have been mutated to alanine alleviates the precocious loss of sister chromatid cohesion and the mitotic arrest of cells lacking shugoshin. These data suggest that shugoshin prevents phosphorylation of cohesin's Scc3-SA2 subunit at centromeres during mitosis. This ensures that cohesin persists at centromeres until activation of separase causes cleavage of its alpha kleisin subunit. Centromeric cohesion is one of the hallmarks of mitotic chromosomes. Our results imply that it is not an intrinsically stable property, because it can easily be destroyed by mitotic kinases, which are kept in check by shugoshin.     

DNA-Replikation und Chromosomenstruktur von Mesostoma (Turbellaria)

During meiosis in M. ehrenbergi (2n=10) and M. lingua (2n=8) male certain chromosomes never pair completely. In these bivalents only terminal pairing appears, crossing over could not be proved by ³H-thymidine autoradiography. DNA amounts of the M. ehrenbergi and M. lingua genomes are in a proportion of 10∶1. The mitotic S-phase of spermatogonia in M. ehrenbergi is twice as long as in M. lingua. In metaphase of spermatogonia a differentiated DNA replication pattern can be identified in M. ehrenbergi as late-pulse-replicating segments. After incorporation of ³H-thymidine X₂-metaphase chromosomes can be found, which show single chromatid labeling, terminal and intercalary isolabeling as well as kinds of chromosome labeling, which can only result from sister strand exchange. After treating the chromosomes with low temperature, colchicine or by hydrolysis (60° C) substructures of the chromatin become visible in both spezies which however are evaluated as artefacts. — Formation of the different isolabeling types is discussed on the basis of a two-strand model of the chromosome fibril. A hypothesis is formulated that the surplusage of DNA in M. ehrenbergi is distributed over all the length of the chromatids as small parts of heterochromatin. This hypothesis is supported by investigations of the DNA replication and the contractility of the chromosomes. Furthermore, a pattern of small DNA particles can be demonstrated after partial destruction of the DNA in metaphase chromosomes of M. ehrenbergi, which could represent this intercalary heterochromatin.     

Normal and BrdC-substituted chromosomes during spermatogenesis with an endomeiotic chromosome reduplication in Carausius morosus Br.

Spermatogenesis involving an additional chromosome reduplication during zygotene in sporadic males and intersexes of the thelytokous phasmid Carausius morosus Br. has been examined using differential staining of chromatids after 5-bromodeoxycytidine incorporation. After reduplication autobivalents are formed by synapsis between identical sister chromosomes. Chiasmata are only formed after reduplication; they do not occur in constitutive heterochromatin, but can be formed in facultative heterochromatin, dependent on heteropycnosis and sex. Quadrivalents and U-type exchanges occur. In spermatogonia and spermatocytes the number of differentially stained chromosomes varies considerably; sister chromatid exchanges hardly appear. Sex bivalents with differentially stained chromosomes have a lower chiasma frequency than normally stained sex bivalents. Bivalents show reduced staining of all four, two outer, or one inner chromatid. Autobivalents arise in the same way as diplochromosomes; chromatids with the oldest DNA sub-units remain together during reduplication and are thus involved in sister chromosome pairing. The additional reduplication begins 7 days after the premeiotic S-phase, first metaphase after 19 days. Spermatogenesis is abnormal from first anaphase onwards.     

A change in sister chromatid behavior precedes nuclear division in Saccharomyces cerevisiae

We used a genetic assay to monitor the behavior of sister chromatids during the cell cycle. We show that the ability to induce sister chromatid exchanges (SCE) with ionizing radiation is maximal in budded cells with undivided nuclei and then decreases prior to nuclear division. SCE can be induced in cells arrested in G2 using either nocodazole or cdc mutants. These data show that sister chromatids have two different states prior to nuclear division. We suggest that the sister chromatids of cir. III, a circular derivative of chromosome III, separate (anaphase A) prior to spindle elongation (anaphase B). Other interpretations are also discussed. SCE can be induced in cdc mutants that arrest in G2 and in nocodazole-treated cells, suggesting that mitotic checkpoints arrest cells prior to sister chromatid separation.     

Contribution of hCAP-D2, a non-SMC subunit of condensin I, to chromosome and chromosomal protein dynamics during mitosis

Condensins are heteropentameric complexes that were first identified as structural components of mitotic chromosomes. They are composed of two SMC (structural maintenance of chromosomes) and three non-SMC subunits. Condensins play a role in the resolution and segregation of sister chromatids during mitosis, as well as in some aspects of mitotic chromosome assembly. Two distinct condensin complexes, condensin I and condensin II, which differ only in their non-SMC subunits, exist. Here, we used an RNA interference approach to deplete hCAP-D2, a non-SMC subunit of condensin I, in HeLa cells. We found that the association of hCAP-H, another non-SMC subunit of condensin I, with mitotic chromosomes depends on the presence of hCAP-D2. Moreover, chromatid axes, as defined by topoisomerase II and hCAP-E localization, are disorganized in the absence of hCAP-D2, and the resolution and segregation of sister chromatids are impaired. In addition, hCAP-D2 depletion affects chromosome alignment in metaphase and delays entry into anaphase. This suggests that condensin I is involved in the correct attachment between chromosome kinetochores and microtubules of the mitotic spindle. These results are discussed relative to the effects of depleting both condensin complexes.     

Investigation of Homologous Crossing over and Sister Chromatid Exchange in the Wheat Nor-B2 Locus Coding for Rrna and Gli-B2 Locus Coding for Gliadins

Recombination was investigated within the Nor-B2 locus of wheat chromosome 6B that contains several thousand of the 18S-5.8S-26S rRNA (rDNA) repeated units. Additionally, recombination was assessed for several chromosome regions, in arm 6Bq between the centromere and the B2 locus (awn suppressor) and in arm 6Bp between the centromere and Nor-B2, between Nor-B2 and a distal C-band and between Nor-B2 and Gli-B2 coding for gliadins. The experimental design permitted the distinction between crossing over between homologous chromosomes and exchange between sister chromatids. No homologous crossing over within the Nor-B2 locus was found in a sample of 446 chromosomes, but one exchange with the attributes of unequal sister chromatid exchange was identified. The molecular characteristics of this presumed sister chromatid exchange indicate that the spacer variants present in the Nor-B2 locus are clustered. No homologous recombination was detected within the distal Gli-B2 locus containing repeated genes coding for gliadin seed-storage proteins. Both arms of chromosome 6B showed low crossing-over frequency in the proximal regions. The distance from the centromere to Nor-B2 was only from 0.3 to 2.2 cM although it accounts for about two-thirds of the metaphase chromosome arm, which shows a great distortion of the metaphase map of the arm. The level of homologous recombination within the Nor-B2 locus is lower than in the chromosome region immediately distal to it. Whether it is comparable to that in the chromosome region proximal to it could not be determined. Recombination frequencies of different pairs of chromosome 6B in all but one interval paralleled the frequencies of their metaphase I pairing: Lower pairing at metaphase I was paralleled by lower crossing-over frequency. This relationship indicated that reduced metaphase I pairing between 6B chromosomes from different populations is due to impaired crossing-over and not due to precocious chiasma terminalization.     

GFP tagging of budding yeast chromosomes reveals that protein–protein interactions can mediate sister chromatid cohesion

Background Precise control of sister chromatid separation is essential for the accurate transmission of genetic information. Sister chromatids must remain linked to each other from the time of DNA replication until the onset of chromosome segregation, when the linkage must be promptly dissolved. Recent studies suggest that the machinery that is responsible for the destruction of mitotic cyclins also degrades proteins that play a role in maintaining sister chromatid linkage, and that this machinery is regulated by the spindle-assembly checkpoint. Studies on these problems in budding yeast are hampered by the inability to resolve its chromosomes by light or electron microscopy.Results We have developed a novel method for visualizing specific DNA sequences in fixed and living budding yeast cells. A tandem array of 256 copies of the Lac operator is integrated at the desired site in the genome and detected by the binding of a green fluorescent protein (GFP)–Lac repressor fusion expressed from the HIS3 promoter. Using this method, we show that sister chromatid segregation precedes the destruction of cyclin B. In mad or bub cells, which lack the spindle-assembly checkpoint, sister chromatid separation can occur in the absence of microtubules. The expression of a tetramerizing form of the GFP–Lac repressor, which can bind Lac operators on two different DNA molecules, can hold sister chromatids together under conditions in which they would normally separate.Conclusions We conclude that sister chromatid separation in budding yeast can occur in the absence of microtubule-dependent forces, and that protein complexes that can bind two different DNA molecules are capable of holding sister chromatids together.