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Chromosome Research 9: 175^198, 2001. # 2001 Kluwer Academic Publishers. Printed in the Netherlands 175 Review A model for chromosome structure during the mitotic and meiotic cell cycles Stephen M. Stack & Lorinda K. Anderson Department of Biology and Cell and Molecular Biology Program, Colorado State University, Fort Collins, CO 80523, USA; Fax: 970-491-0649; Tel: 970-491-6802; E-mail: [email protected] Received 12 October 2000; received in revised form and accepted for publication by H. Macgregor 2 January 2001 Key words: chiasmata, chromosome cores, chromosome structure, coiling, crossing over, meiosis, mitosis, model, presynaptic alignment Abstract The chromosome scaffold model in which loops of chromatin are attached to a central, coiled chromosome core (scaffold) is the current paradigm for chromosome structure. Here we present a modi¢ed version of the chromosome scaffold model to describe chromosome structure and behavior through the mitotic and meiotic cell cycles. We suggest that a salient feature of chromosome structure is established during DNA replication when sister loops of DNA extend in opposite directions from replication sites on nuclear matrix strands. This orientation is maintained into prophase when the nuclear matrix strand is converted into two closely associated sister chromatid cores with sister DNA loops extending in opposite directions. We propose that chromatid cores are contractile and show, using a physical model, that contraction of cores during late prophase can result in coiled chromatids. Coiling accounts for the majority of chromosome shortening that is needed to separate sister chromatids within the con¢nes of a cell. In early prophase I of meiosis, the orientation of sister DNA loops in opposite directions from axial elements assures that DNA loops interact preferentially with homologous DNA loops rather than with sister DNA loops. In this context, we propose a bar code model for homologous presynaptic chromosome alignment that involves weak paranemic interactions of homologous DNA loops. Opposite orientation of sister loops also suppresses crossing over between sister chromatids in favor of crossing over between homologous non-sister chromatids. After crossing over is completed in pachytene and the synaptonemal complex breaks down in early diplotene ( diffuse stage), new contractile cores are laid down along each chromatid. These chromatid cores are comparable to the chromatid cores in mitotic prophase chromosomes. As an aside, we propose that leptotene through early diplotene represent the `missing' G2 period of the premeiotic interphase. The new chromosome cores, along with sister chromatid cohesion, stabilize chiasmata. Contraction of cores in late diplotene causes chromosomes to coil in a con¢guration that encourages subsequent syntelic orientation of sister kinetochores and amphitelic orientation of homologous kinetochore pairs on the spindle at metaphase I. Introduction In the 1950s, the general model for chromosome structure included a coiled chromosome core embedded in a matrix that is covered by a thin pellicle (e.g. Schrader 1953, DeRobertis et al. 1954, see Ohnuki 1968 for a review). However, electron microscopic observations in the 1960s were interpreted to indicate that chromosomes consist of a dense tangle of 10^30-nm chromatin 176 ¢bers with neither a core to the inside nor a pellicle to the outside, i.e. the `folded-¢ber model' (e.g. DuPraw 1968, and see Belmont & Bruce 1994 for a newer version of this model). The existence of coiling in some chromosomes was acknowledged but the only recognized chromosome cores were axial elements and lateral elements related to the synaptonemal complex (SC; see Moses 1968 for a contemporary review). Subsequently, new observations and experiments resulted in a new paradigm for chromosome structure (the chromosome scaffold model) that in many ways resembles the preelectron microscope chromosome model from the 1950s: Condensed chromosomes are coiled (e.g. Ohnuki 1968), and each chromatid has a scaffold (core) of non-histone proteins running along its length (e.g. Paulson & Laemmli 1977, Earnshaw & Laemmli 1983) that is also coiled (Rattner and Lin 1985, Boy de la Tour & Laemmli 1988). The single most abundant protein component that has been identi¢ed so far in the scaffold is topoisomerase II, an enzyme that has the potential to decatenate tangled DNA by passing one double helix through the other (Berrios et al. 1985, Earnshaw & Heck 1985, Gasser et al. 1986). Loops of DNA (associated with protein to form chromatin matrix in the old terminology) extend laterally from the scaffold (e.g. Gasser & Laemmli 1987, Earnshaw 1988). The bases of DNA loops are bound to chromosome scaffolds by SARs (scaffold attachment regions) that are AT-rich segments of bent DNA to which one or more scaffold proteins bind (e.g. Homberger 1989, von Kries et al. 1991). SARs are also attached to the nuclear matrix during interphase when SARs are called MARs (matrix attachment regions; Mirkovitch et al. 1988, Boulikas 1995). Chromosome scaffolds and nuclear matrix strands appear to be related structures that share some proteins, including topoisomerase II and p62 (e.g. Gasser et al. 1989, Berezney et al. 1995). In this regard, many SARs include the 15-bp consensus sequence for topoisomerase II, indicating that SARs are sites where DNA catenations are resolved (Gasser et al. 1989, Gimënez-Abiän et al. 1995). Finally, chromosomes are surrounded by an RNA-protein S. M. Stack & L. K. Anderson coat ( pellicle in the old terminology) that largely derives from the disassembly of the nucleolus and application of some nucleolar components to condensed chromosomes during mitosis (e.g. Moyne & Garrido 1976, see Hernandez-Verdun & Gautier 1994 for a review). During interphase, the nuclear matrix is probably the counterpart of the chromosome scaffold of mitotic chromosomes (see Gasser et al. 1989 for a review). Newly synthesized DNA and RNA, as well as enzymes involved in these syntheses, are attached to the nuclear matrix. This suggests that loops of DNA are reeled through relatively stationary replication and transcription complexes on the nuclear matrix (see Cook 1999 for a review). Replication origins occur roughly every 15^30 mm of DNA, although the range of replicon size is probably enormous (Gasser et al. 1989, Boulikas 1995, Berezney et al. 2000). Starting from a replication origin, two replication forks proceed in opposite directions until they encounter replication forks advancing from adjacent replicons (Huberman & Riggs 1968, Jackson & Pombo 1998). Clusters of synchronous replicons can be labeled and observed as £uorescent foci by light microscopy or as electron-dense masses of chromatin by electron microscopy (Nakayasu & Berezney 1989, Hozäk et al. 1993, Newport & Yan 1996). If the association of DNA with the matrix were mediated by replication complexes only, then one would expect a changing sample of the DNA to be associated with the nuclear matrix during S phase. However, Gasser et al. (1989) reported that MARs are associated with the matrix/scaffold throughout the cell cycle, so this subset of DNA never leaves the nuclear matrix/scaffold. Assuming that these descriptions of chromosome structure and DNA replication are generally accurate, here we present an extension of these ideas to describe a model for some aspects of the structure and behavior of chromosomes through the mitotic and meiotic cell cycles. In the descriptions that follow, ideas that we view as more speculative are in italics. See Earnshaw (1988), Manuelidis & Chen (1990), Cook (1995), and Heslop-Harrison (2000) for reviews and other models for chromosome structure. Chromosome structure through mitosis and meiosis Model for the structure of chromosomes through the mitotic cell cycle G1 phase During the G1 phase, each chromosome consists of only one chromatid that relaxes and uncoils (Belmont & Bruce 1994). Each chromatid carries a single DNA double helix that is supercoiled around clusters of histones to form nucleosomes (Kornberg 1974, Olins & Olins 1974). Each nucleosome consists of two molecules each of histone H2A, H2B, H3, and H4. Tandem nucleosomes form a strand of chromatin with a diameter of 10 nm. Through interaction with H1 histones, these 10-nm ¢laments are often supercoiled to form 30 nm chromatin ¢bers (Finch & Klug 1976, Losa et al. 1984, but see Woodcock & Horowitz 1991 who present evidence for a different organization of nucleosomes in transcriptionally active cells). The 30-nm chromatin ¢bers form loops attached at their bases (via MARs, replication origins, and promoters) to strands of nuclear matrix consisting of protein and RNA (Nickerson et al. 1995, Boulikas 1995, Jackson et al. 1996, Ma et al. 1999, see Cremer et al. 1995 for different models of the nuclear matrix). Loops of DNA (chromatin) extend from only one side of a matrix strand in G1 (Figures 1, 12A). (The basis for assuming this arrangement of loops is explained later.) These nuclear matrix strands to which chromatin loops are attached were derived from single, relaxed and extended chromatid cores from the previous anaphase (Figure 1, 12A), which show relic coiling from the previous anaphase (e.g. Vejdovsk 1911^1912 for early illustrations, Manuelidis & Chen 1990, Belmont & Bruce 1994). Interconversion of nuclear matrix strands and chromosome cores is suggested by premature chromosome condensation experiments in which G1 cells are fused with metaphase cells (e.g. Rao & Johnson 1970). While G1 nuclear matrix strands from unfused cells do not stain with silver, each long prematurely condensed G1 chromosome develops a single silver-stainable core throughout its length (Gimënez-Abiän et al. 1995). This core is comparable to the single silver-stained core in each chromatid of metaphase and anaphase chromosomes (Gimënez-Abiän et al. 1995). Acquisition 177 of silver stainability must be due to addition of new proteins and/or modi¢cation of existing proteins that takes place during prophase. The relationship between matrix strands and/or chromosome cores is further supported by reports that DNA loops are attached to matrix strands and chromosome cores by the same AT-rich sequences (MARs and SARs ^ Mirkovitch et al. 1984, Gasser et al. 1986) and by reports that chromosome cores and nuclear matrix strands share some of the same proteins (Detke & Keller 1982, Peters et al. 1982, Gasser et al. 1989), probably including RNA polymerases I and II and lamins (Matsui et al. 1979, Jackson & Cook 1995). S phase During early S phase in mammalian cells, groups of approximately 40 adjacent replicons initiate replication together in electron-dense bodies 100^200 nm in diameter (replication factories) attached to the nuclear matrix (e.g. Jackson & Cook 1995). MARs are attached to the nuclear matrix throughout interphase, and pulse labeling during S-phase demonstrates that nascent DNA is closely associated with the nuclear matrix as well (e.g. Pardoll et al.1980, Jackson & Pombo 1998). Thus, at the beginning of S phase, replication origins must be attached to the nuclear matrix at the centers of replicons (Figure 2), and MARs may reside at or near replication origins (see Boulikas 1995 for a review). When replication is initiated at the base of a DNA loop, the two replication forks remain at the replication complex, and parental DNA is pulled in from the left and right (Figures 3^5) (Pardoll et al. 1980). Nucleosomes are disassembled at replication forks before their histones reassociate (along with new histones) with replicated DNA to form new nucleosomes in the daughter strands of chromatin (e.g. Randall & Kelly 1992). Replication origins and MARs are duplicated ¢rst and quickly reattach to the matrix strand, i.e. replication origins and MARs do not emerge from the matrix strand on daughter DNA loops (Cook 1991). However, as more parental DNA without MARs is reeled in from adjacent parental loops, two sister loops of DNA emerge from the left and two sister loops of DNA emerge from the right of the replication complex. The nascent sister 178 loops emerge at 180 to each other. Eventually replication forks from adjacent replicons meet. At this point, the DNA is released from replication complexes and matrix strands except at MARs (Figure 6). This model is in agreement with observations that sister DNA sequences are separated from each other by the time replication is completed (Selig et al. 1992). There is some tangling (catenation) of sister DNA loops but this is minimized by orientation of sister loops in opposite directions with the result that most of the tangling should be near the bases of the loops. Also, MARs often include the 15-bp consensus sequence for topoisomerase II (Gasser et al. 1986), and topoisomerase in the matrix strands is in the right position to decatenate sister loops of DNA when they come under tension as the sister chromatids begin to shorten and separate in prophase (Earnshaw & Heck 1985, Woessner et al. 1987, Saitoh & Laemmli 1994, Gimënez-Abiän et al. 1995). It is also during S phase when cohesin complexes establish linkage between sister chromatids (Uhlmann & Nasmyth 1988, Nasmyth et al. 2000). Since sister chromatid cohesion can be established only during S phase, cohesion complexes probably have to be inserted when the bases of sister DNA loops are close together during or right after replication. A number of observations indicate that during S phase there is a major reorganization of chromosome structure. At this time, prematurely condensed chromosomes take on a `pulverized' appearance (e.g. Sperling & Rao 1974), silverstained cores of prematurely condensed chromosomes show discontinuities (Gimënez-Abiän et al. 1995), and interphase chromosomes lose distinct borders (Stack et al. 1977, Brown et al. 1978). These observations are probably related to local dissolution of the single G1 matrix strands during DNA replication. Groups of adjacent replicons initiate replication together in factories (e.g. Hozäk et al. 1993, Jackson & Cook 1995), indicating coordination of replication units related to chromosome structure that is more or less maintained even during S phase (Manuelidis 1990). As batteries of replicons complete replication, a new matrix strand forms in association with the MARs of one DNA double helix (chromatid), and another matrix strand forms in association with the MARs of the sister DNA double helix. Sister matrix S. M. Stack & L. K. Anderson strands are in close association. These segments of sister matrix strands link up progressively as batteries complete replication until a continuous pair of sister matrix strands extends throughout the length of a duplicated chromosome at the end of S phase, i.e. the beginning of G2 (Figure 6 & 7; e.g. Gimënez-Abiän et al. 1995). How sister matrix strands form in close association with each other and tell one sister DNA strand from the other remains to be explained, but it may involve opposite orientation of sister DNA loops. G2 phase During G2, chromatin is diffuse, consisting of 10and 30-nm-diameter chromatin ¢bers. Sister DNA (chromatin) loops extend in opposite directions from duplicated but closely associated sister matrix strands (Figure 7). A G2 chromosome occupies a cylinder of space in the nucleus. DNA loops of each sister chromatid probably occupy a half cylinder of space with sister nuclear matrix cores in the center of the cylinder of chromatin (Figure 7C). Loops of DNA on one chromatid can extend from the matrix core outward anywhere within the half cylinder occupied by one chromatid, with the caveat that the sister loop in the sister chromatid extends from its matrix core in the opposite direction within the con¢nes of its own half cylinder. Matrix strands are £exible, and DNA loops are not condensed, so even though sister loops extend in opposite directions, sister DNA loops are able to come in contact and act as preferred templates (compared to homologous chromosomes) for repair of DNA damage during S and G2 (van Heemst & Heyting 2000). Consistent with this model, premature chromosome condensation of G2 nuclei results in the appearance of elongate chromosomes that consist of two parallel sister chromatids (e.g. Rao & Johnson 1970). Each prematurely condensed chromatid has a silverstainable core, and sister cores are closely associated (Gimënez-Abiän et al. 1995, 2000). See Cook (1991) for a very similar model for interphase chromosome structure and replication, except for the stipulation that sister DNA loops extend in opposite directions from the duplicated matrix strand. Chromosome structure through mitosis and meiosis 179 Figures 1^11 (overleaf). Diagrammatic representations of chromosome structure through the mitotic cell cycle. Drawings are not to scale. Telophase/G1 . (A) Longitudinal view of a segment of a decondensing chromosome during telophase or a decondensed chromosome during G1. The anaphase chromosome core (copper-colored) relaxes and uncoils as it is converted by modi¢cation of its proteins into a matrix strand during telophase. DNA loops (green lines ^ mostly 30-nm diameter chromatin ¢bers) are attached to the core/matrix strand via MARS (green dots) with closely associated replication origins (included in green dots). Probable attachments to transcription complexes are not illustrated for simplicity (Jackson et al. 1996). The DNA loops are present on only one side of the matrix strand. For diagrammatic purposes, the loops are shown the same length, in one plane, and short relative to the core/matrix strand. Also see Figure 12A. (B) Cross section of a core/matrix strand with one loop extending upward. (C) End view of a segment of a core/matrix strand with successive DNA loops extending in various directions within a half-circle (or within a half-cylinder three-dimensionally). This view is not shown again until Figure 7C. Figure 2. Late G1 . (A) Longitudinal view of a nuclear matrix strand that includes replication complexes (gray ovals) and MARs (near replication origins) at the bases of DNA loops. (B) Cross section of a matrix strand showing only one loop. Figure 3. Early S-phase. (A) Longitudinal view of a matrix strand during DNA synthesis. The matrix strand is partly disassembled (note that the copper-colored strand is missing) at sites actively undergoing DNA replication. In this string of replication complexes, replication origins and MARS are the ¢rst sequences to be replicated, but they remain associated with the replication complexes, as do replication forks. Adjacent replicons tend to initiate and complete replication together in groups associated with dense structures that have been referred to as replication factories (e.g. Hozäk et al. 1993). (B) Cross section of a replication complex showing two MARs/replication origins as well as an unreplicated loop extending upward. Figure 4. Mid S-phase. (A) Longitudinal view of a string of replication complexes with newly formed sister loops of DNA extending upward and downward (in opposite directions) from their bases in replication complexes. For diagrammatic purposes, the unreplicated (parental) DNA is shown in the same plane as the replicated sister loops but the parental DNA may be at a 90 angle to both newly replicated sister loops. The loops of unreplicated DNA shorten as they are drawn into replication complexes and fed into replication forks. (B) Cross section of a replication complex showing one long unreplicated loop (above) and shorter replicated loops above and below. Figure 5. Late S-phase. (A) Longitudinal view of a string of replication complexes near the end of DNA synthesis. Parental DNA shortens as replicated DNA loops continue to enlarge. (B) Cross section showing sister loops extending above and below a replication complex. Figure 6. DNA synthesis complete. (A) Longitudinal view of a string of replication complexes after the last segments of DNA are replicated between adjacent replication complexes (adjacent replication forks meet). Completely replicated sister loops of DNA are released from the replication complexes except at MAR/replication origins. (B) Cross section showing sister loops extending in opposite directions from each other and attached to replication complexes at MAR/replication origins. Figure 7. G 2 . (A) Longitudinal view of assembling sister matrix strands (light copper) after disassembly of replication complexes. Sister DNA loops extend in opposite directions from adjacent sister cores. Each newly formed matrix strand is associated with MARs on one DNA double helix. Also see Figure 12B. (B) Cross section of forming sister matrix strands/cores with two sister loops of DNA extending in opposite directions. (C) End view of a segment of matrix strand showing sister DNA loops (same color) extending in opposite directions from the duplicated matrix strands/cores. Loops from one chromatid are found within one of the half circles (e.g. upper gray half circle or half cylinder three-dimensionally), and these loops from one chromatid do not overlap with DNA loops from the sister chromatid in the other (lower gray) half circle. Figure 8. Early prophase. (A) Longitudinal view of early prophase chromosome. What were formerly nuclear matrix strands are being modi¢ed by changes in their proteins to become more substantial chromatid cores ( scaffolds, copper) that are capable of active shortening. Thirty-nm chromatin loops collapse toward centrally located sister chromatid cores. The loops are connected to chromatid cores by SARs ( interphase MARs). Sister chromatids and sister cores are held together throughout their length by DNA catenations and cohesins. Also see Figure 12B. (B) Cross section showing two condensed sister chromatin loops extending in opposite directions from adjacent sister chromatid cores. Figure 9. Late prophase. Longitudinal view of contracting (actively shortening) chromatid cores. Contraction results in some shortening of prophase chromosomes. However, as long as sister cores are trapped together in the center of the chromatin mass, neither extensive shortening nor coiling of chromatids is possible. In this diagram, the SARS are somewhat closer together than in Figure 8 due to contraction of the sister cores. Also see Figure 12C. Figure 10. Prometaphase/metaphase. Three-dimensional drawing (at lower magni¢cation than the diagrams in Figures 1^9). Sister chromatid cohesion has relaxed along the length of chromosomes except at centromeres. Whatever the nature of sister chromatid cohesion, sister chromatids remain close to each other along their length. Active shortening of chromatid cores causes chromatid arms to coil and shorten. Coiling does not occur at the centromere because the two chromatid cores are still held close together at this location. Because centromeres are not coiled, they look like constrictions of the chromosome, i.e. primary constrictions. Kinetochores (yellow) face in opposite directions and attach to kinetochore microtubules extending from opposite poles. At the end of each chromatid, individual chromatin loops coming off one side of each chromatid core are illustrated. To the right in a transparent view, the coils of both chromatids are loosened and the paths of the chromatid cores are shown. To the left, note a reversal of coiling direction in the upper chromatid arm. However, sister chromatids usually coil as mirror images of each other. Also see Figure 12D. Figure 11. Anaphase. Three- dimensional drawing. Sister chromatid cohesion has been released at the centromere region, and this permits coiling to extend through centromeres. The two sister chromosomes (chromatids) are moving toward opposite poles. A reversal of coiling is visible in the upper left chromosome (see Figures 10 & 12E). Within each chromosome, the contracted chromosome core (copper) remains to one side of the chromatin. To the right in a transparent view, the coils are loosened and the paths of the chromatid cores are shown. 180 S. M. Stack & L. K. Anderson Chromosome structure through mitosis and meiosis 181 182 Figure 12. Rope and elastic models of mitotic chromosomes. The chromatin of each chromatid is represented by a segment of £exible white rope that was initially 53 cm long in all of the models. The chromatid core is represented by a black elastic band that was sewn to the white rope. Kinetochores are represented by felt patches labeled with `K.' In these models, 1 cm represents 1 mm, i.e. the ropes are 10 000 : 1 scale models of chromosomes. (A) G1 chromosome. Here the elastic band S. M. Stack & L. K. Anderson was not stretched before it was sewn to the rope because at this stage the chromosomes are decondensed and the cores are not contracted. The model is 53 cm long. Also see Figures 1 & 2. (B) G2 early prophase chromosome. Two ropes like those in A (above) were sewn together with the two (non-stretched) elastic bands in the center. The bands between the ropes (arrow) represent the chromatid cores between sister chromatids held together by cohesins. The model is 53 cm long. Also see Figure 7. (C) Prophase chromosome. Here, a 53 cm length of white rope was sewn to a maximally stretched elastic band 53 cm long. If not attached to the rope, this stretched elastic band will contract to about 22 cm (two-fold) upon release of the tension. A second 53-cm rope and stretched elastic band was prepared the same way. Then, while fully stretched to 53 cm, the two ropes were sewn to each other lengthwise with the stretched elastic bands adjacent between them (arrow). When tension on this composite of ropes and stretched elastic is released (as shown), the length decreases from 53 cm to 40 cm (a 32% reduction in length) with no tendency to coil. This suggests that only modest shortening of a chromosome can be achieved by a contractile core located between two closely associated chromatids. Also see Figures 8 & 9. (D) Metaphase chromosome. Two ropes attached to bands of stretched elastic were sewn together as in C above. Subsequently, the threads holding the ropes together distal to the centromeres were cut to free the ropes (chromatid arms) from each other. The response of the relatively incompressible rope (representing chromatin) attached on one side to a contractile band (the chromosome core) is to coil with the contractile band taking the shorter, inside track. These coils of rope are 15 cm long, representing a 72% reduction in length compared with G2/early prophase chromosomes (see B above), demonstrating that coiling achieves much more shortening than would be anticipated by simple contraction of the chromatid cores (see C above). Coiling occurs everywhere except at the centromere (= primary constriction) where sister chromatids are still bound to each other. The arrowhead points to a reversal of coiling. To the left, the bottom chromatid has been pulled out so that the inner black elastic band can be seen. Also see Figure 10. (E) Anaphase chromosomes. A metaphase chromosome model was prepared as described in D above. To mimic the loss of sister chromatid cohesion from centromeres at the beginning of anaphase, the threads holding the two ropes together at the centromere region were removed with the result that both ropes coiled along their entire length. The two ropes were then oriented to represent their relationship in mid-anaphase. These coils are about 11 cm long, representing a reduction in length of 79% compared with G2-early prophase chromosomes (see B above). Thus, the majority of reduction in length of mitotic chromosomes is due to coiling rather than contraction per se. The arrowhead points to a reversal of coiling. Also see Figure 11. Chromosome structure through mitosis and meiosis Mitosis As cells pass into prophase, any remaining 10-nm chromatin ¢bers are supercoiled to form 30-nm ¢bers, and most transcription ceases, related to deacetylation of histones and loss of transcription factors from chromatin (Mart|¨ nez-Balbäs et al. 1995 and references therein). Thirty-nm ¢bers are pulled toward their attachment sites on cores by protein complexes called condensins (see Hirano 1999 for a review). At the same time, nuclear matrix strands are converted into more substantial chromatid cores by adding proteins and/or modifying proteins of the matrix strand that now can be silver-stained. At the initiation of chromosome condensation in prophase, sister chromatid cores are close together as observed in prematurely condensed G2 chromosomes and silver-stained prometaphase chromosomes (Figures 7, 8 & 12B; Gimënez-Abiän et al. 1995, 2000). Chromosomes shorten during prophase (Figure 9). Shortening is initially due to active lengthwise contraction of the paired sister cores between as-yet unseparated chromatids that are held together by DNA catenations and cohesin complexes. Shortening of cores is inhibited to a degree by their location in the interior of the mass of sister chromatin that resists being packed more tightly (see the rope and elastic model shown in Figure 12C). Sister chromatid cohesion is mediated by cohesins (see Orr-Weaver 1999 for a review) and catenations (tangling and interlocking) of DNA loops. Catenation of sister DNA loops is most likely at their bases in cores where topoisomerase II is available to resolve interlocking. Resolution probably occurs when catenations are brought under tension by chromatin condensation and contraction of cores during prophase (Downes et al. 1991, Gorbsky 1994, Gimënez-Abiän et al. 1995). If topoisomerase II is inhibited and catenations cannot be resolved, silver staining reveals intimately paired sister cores embedded in extended chromosomes that persist long after sister cores normally would have separated in prometaphase (Gimënez-Abiän et al. 1995, 2000). This is strong evidence that sister cores are located together and to one side of sister chromatids (Figures 7^9, 12B, C). Without decatenation, full chromosome condensation 183 and sister chromatid separation is not possible (Uemura et al. 1987, Newport & Spann 1987, Adachi et al. 1991, Downes et al. 1991, 1994, Gimënez-Abiän et al. 1995, 2000). At the transition from prophase to prometaphase, sister chromatid arms separate to some degree as catenations are eliminated and cohesins break down. This permits sister chromatid arms and their cores to separate and coil (GimënezAbiän et al. 1995, 2000, see Manton 1950 for a review of chromosome coiling; Figures 10, 12D). Coiling is important because it is the primary means by which chromosomes are compacted and shortened enough to be separated from each other within the con¢nes of the cell (Figure 12). Sister chromatids usually show mirror image patterns of coiling (Boy de la Tour & Laemmli 1988) that is probably related to their identical chromatin structure. This relationship is supported by the observations of Baumgartner et al. (1991) that single copy sequences usually occupy mirror image positions on sister chromatids in human chromosomes. Because the position of single copy sequences usually remained the same on chromosomes that varied somewhat in length, they concluded that ¢nal chromosome condensation is due to tighter packing of the coils rather than continued coiling. Chromatin structure affects the number and placement of coils based on evidence that the coiling pattern of individual mammalian chromosomes is related to their chromomere and G-band patterns (e.g. Kato & Yosida 1972, Okada & Comings 1974, Harrison et al. 1981). However, sister chromatids can differ from each other by reversals in their coiling pattern (Figures 10, 11 & 12D, Manton 1950, Ohnuki 1968). There have been a number of different (often con£icting) proposals to explain coiling (see John & Lewis 1965 for a review). In our model, the initial location of a chromosome core along one side of a chromatid suggests a demonstrable physical mechanism for coiling (see illustrations of the rope and elastic chromosome model in Figure 12). The model is based on the behavior of a £exible rod ( rope) attached along its length to a strip of contractile material ( stretched elastic). When the strip contracts, the whole structure spirals with the contractile strip taking the shorter route along the inside of the spiral and the £exible rod taking 184 the longer route to the outside of the spiral. While an elastic strip has been used to prepare the model, we are not suggesting that the core is elastic but that it is contractile. Indeed, Gimënez-Abiän et al. (1995) have shown that chromatid cores shorten in length by almost two-fold from early prometaphase to metaphase. Gimënez-Abiän et al. (1995) and Nokkala & Nokkala (1984) also illustrate that each chromatid core is in a lateral position on each sister chromatid from prophase through prometaphase but they do not suggest that shortening cores drives coiling. In regard to this model, it is interesting that contraction and coiling of the spasmoneme of Vorticella looks like it uses the proposed mechanism for chromosome coiling (see Mahadevan & Matsudaira 2000 for contraction of the spasmoneme, although again they do not suggest this model for coiling; see the cover of Science Vol. 288, No. 5463, April 7, 2000). As the model is described, one might expect a hollow (chromatinless) center in each contracted chromatid, but generally hollow centers are not observed in aldehyde-¢xed and sectioned chromosomes (Manuelidis & Chen 1990). However, if contraction is strong enough, the twisted core simply takes up a central position in the coil without any central open space. With the elimination of a central space, there is little or no change in the number of gyres but there is a slight decrease in diameter of the coiled chromatid. We have demonstrated this with a rope and elastic model (not illustrated). Techniques that demonstrate coiling usually require a treatment that swells the chromatin matrix to separate the gyres of the chromatid cores (e.g. Matsuura 1938, Ohnuki 1968, Rattner & Lin 1985). At centromeres the two sister chromatids are held together by a special set of proteins (Figures 10 & 12D; see Rieder & Cole 1999 for a review of proteins involved in chromatid cohesion). As long as the contractile sister cores remain in intimate association in the center of the chromatin at the centromere, some shortening can occur here but no coiling. DNA in centromeres is enriched in SARs, so DNA loops in centromeres are short (Strissel et al. 1996). Since sister chromatids are usually mirror images of each other, this means that sister kinetochores forming on these short DNA loops face outward, and kinetochores facing S. M. Stack & L. K. Anderson in opposite directions are closely anchored to the underlying cores (Zhao et al. 2000). In contrast to active centromeres, inactive or dormant centromeres do not form constrictions (e.g. Earnshaw & Migeon 1985 and references therein), suggesting that inactive centromeres do not have the special proteins that hold active centromeres together so inactive centromeres are free to coil. In reference to secondary constrictions, many of the proteins involved in rRNA transcription and processing remain in association with rDNA at NORs throughout mitosis (Jackson & Cook 1995), and at least some of these proteins can be selectively silver stained (e.g. Goodpasture & Bloom 1975, Schubert 1984). However, unlike active centromeres, sister NORs do not show any particular tendency to remain associated when sister chromatid arms begin to condense and coil. We propose that the accumulation of proteins at active NORs interferes with contraction of the core and coiling, and this results in secondary constrictions. Of course, just as in the case of `primary constrictions' at centromeres, `secondary constrictions' are not really constrictions of chromosomes but rather thinner regions of the chromosome due to a lack of coiling. NORs that were not active in the previous interphase lack most proteins involved in rRNA synthesis and processing so they are free to coil and do not form secondary constrictions (e.g. McClintock 1934, Tanaka & Terasaka 1972, Jordan & Luck 1976). Cohesins hold centromeres of sister chromatids together until sister chromatids are completely separated by proteolysis at the metaphase/ anaphase transition (see Rieder & Cole 1999 and van Heemst & Heyting 2000 for reviews). At this time, sister centromeres separate, and primary constrictions disappear (Sumner 1991), indicating that coiling now extends through centromeres as sister chromosomes are drawn to opposite poles during anaphase (Figures 11 & 12E). When the nuclear envelope reforms during telophase, the contracted and coiled chromosome cores relax. The 30-nm-diameter chromatin ¢bers that were held near the chromosome cores are released, except at their bases, to form extended loops. Proteins are lost or exchanged from chromosome cores that now become nuclear matrix strands which no longer stain with silver. Chromosome structure through mitosis and meiosis In short, chromosomes revert to an interphase chromosome structure characteristic of G1 (Figure 1). Model for the structure of chromosomes through the meiotic cell cycle [See Zickler & Kleckner 1998, 1999 for recent reviews of meiosis.] Premeiotic interphase, G1 and S While the events of premeiotic interphase must be fundamentally similar to mitotic interphase, premeiotic interphase lasts much longer (Kofman-Alfaro & Chandley 1970, Bennett 1977, Holm 1977). Because leptotene chromosomes that appear after S phase are exceptionally long, extra time may be needed for establishing this special chromosome organization. For example, variants of H1 histones like meiotin 1 are added to plant primary microsporocyte nuclei (Qureshi & Hasenkampf 1995) and H1t is added to mammalian primary spermatocyte nuclei (Moens 1995). More recently, it has been shown in Coprinus cinereus that proteins known to be involved in forming double strand breaks during prophase I also seem to function in premeiotic S phase where they somehow prepare chromosomes for synapsis and crossing over (Merino et al. 2000, also see Borde et al. 2000). In addition, SARs which were not utilized during the mitotic cell cycle may be enlisted during the meiotic cell cycle, thereby yielding a longer chromosome with shorter DNA loops, and SARs may associate with new and/or additional proteins in matrix strands. In any case, at the end of premeiotic S phase, sister DNA loops extend in opposite directions from duplicated nuclear matrix strands just as at the end of mitotic S phase (compare Figures 7 & 13). Leptotene With little, if any, delay between the end of S phase and the beginning of leptotene (e.g Crone et al. 1965, Callan & Taylor 1968, King 1970, Stern et al. 1975), matrix strands between sister DNA loops are reinforced with additional meiosis- 185 speci¢c proteins (Offenberg et al. 1998) to form axial elements (cores) from which contracting sister DNA (chromatin) loops extend in opposite directions (Figure 13A, B). An axial element probably consists of two intimately associated, parallel sister cores (Nebel & Coulon 1962, Dresser & Moses 1980, and see Zickler & Kleckner 1999 p. 649 for other references), although usually the double nature of axial elements is not obvious (see Dawe et al. 1994 and references therein for examples of visibly double leptotene chromosomes). Since leptotene chromosomes are longer than at any other time during meiosis (except lampbrush chromosomes), the axial core is probably a comparatively rigid uncoiled core at this time (Scherthan et al. 1998). In Bombyx mori and Lilium longi£orum at early prophase I, chromatin ¢bers have been reported to be from 20^30 nm in diameter, which is probably typical of most chromatin ¢bers during interphase and mitosis (e.g. Ris & Kubai 1970, Rattner & Hamkalo 1979, Rattner et al. 1980). However, unlike mitotic chromosomes, there is transcription on the loops of early prophase I chromosomes (Kierszenbaum & Tres 1974, Rattner et al. 1980, Cook 1997 and references therein), suggesting that DNA in chromatin loops during early prophase I is fairly accessible. The importance of an open chromatin conformation for recombination is suggested by observations in S. cerevisiae that hot spots for double strand breaks and recombination are often located in or near promoter regions (whether functional or not). Promoters are known to be sites where DNA is exposed and accessible, and experimental disruption of nucleosome structure leads to double strand breaks in the exposed DNA (e.g. Wu & Lichten 1994). During late leptotene (if not before) and early zygotene, homologous chromosomes are actively moved in the nucleus, and this probably helps homologs come in contact so they can recognize each other and align (pair) in preparation for synapsis (SC formation) (e.g. Parvinen & SÎderstrÎm 1976, Loidl 1993, Dawe et al. 1994, Scherthan et al. 1996, 1998, but see Schwarzacher 1997 for association of homologous arms during premeiotic interphase in wheat). Initial recognition involves weak, homologous, paranemic DNA/DNA interactions that are stable only when reinforced by 186 S. M. Stack & L. K. Anderson Figures 13^19. Diagrammatic representations of meiotic chromosome structure from leptotene through anaphase I. See Figures 1^6 for diagrams of chromosome structures that are the same for mitotic and premeiotic interphase. Drawings are not to scale. Chromosome structure through mitosis and meiosis other nearby interactions (Figure 13B; e.g. McKee et al. 1992, Kleckner & Weiner 1993, CameriniOtero & Hsieh 1993, Weiner & Kleckner 1994, Gaillard & Strauss 1994, see Cook 1997 for other references and a model for pairing based on transcription). Breaks in DNA are not required for these weak interactions, and the interactions are not directly involved in crossing over, only homologous pairing. Presynaptic alignment (pairing) is only possible between homologs with the same chromatin pattern of exposed DNA loops (Rocco & Nicolas 1996, also see Karpen et al. 1996). This is analogous to aligning unique bar codes on commercial product labels (see Figure 20 for a descrip- 187 tion of the bar code model and the role of the bouquet orientation in presynaptic alignment). It is important that sister loops are contracted and reside on opposite sides of chromosomes to avoid saturating homologous DNA^DNA interactions with interactions between sister loops of DNA. In support of this idea, leptotene through pachytene is the only period in the life cycle of Saccharomyces cerevisiae when chromatin is reported to be contracted (Dresser & Giroux 1988). A related observation involves a 1.7-Mb lambda transgene that forms a long loop extending from a mouse SC where its base is anchored (Moens et al. 1997b). Presumably, the long loop is due Figure 13. Leptotene. (A) Longitudinal view of a segment of a leptotene chromosome after DNA replication is complete (see Figure 6). Proteinaceous axial elements (red^violet) assemble in association with MARs (green dots) at the base of sister DNA loops. Although not usually obvious, each axial element consists of two closely associated sister elements. Chromatin condenses by collapsing 30 nm diameter chromatin loops toward axial elements. The use of more MARs may additionally shorten chromatin loop lengths. Sister DNA loops are represented in the same color but on opposite sides of the axial element. (B) End view of segments of two homologous leptotene chromosomes. For each chromosome, sister DNA loops (same color) extend from axial elements in opposite directions. Homologous DNA loops are shown in different shades of the same color. For example, the black and gray loops are homologous as are the light green and dark green loops. Because the chromatin loops have condensed, it is dif¢cult for sister chromatin loops to contact one another, whereas homologous loops are free to associate. Here, (green) homologous DNA loops have made contact. Such contacts mediate presynaptic alignment. Figure 14. Zygotene/pachytene. End view of a segment of synaptonemal complex (SC). SCs form during zygotene^pachytene. When incorporated into SCs, axial elements are renamed lateral elements (red^violet). Each lateral element is shown divided into two sister elements. Occasionally a lateral element twists so the lower sister element (as viewed here) moves to the upper side and the upper sister element moves to the lower side (not shown). Twisting of lateral elements relative to one another is likely because leptotene chromosomes have been reported to be twisted (Oehlkers & Eberle 1957). This accounts for observations that either sister in each homolog may be involved in a crossover. During SC formation, sister DNA loops (shown the same color) are de£ected somewhat from a strictly opposite arrangement around lateral elements (LEs) to make room for transverse ¢laments in the central region of the SC. Even so, sister loops are sterically inhibited from coming into contact with each other. On the other hand, homologous loops (loops of similar colors, e.g. light green and dark green loops) are in close association and free to interact for repair and recombination. Here a recombination nodule mediates a crossover between the two homologous green loops. Figure 15. Pachytene. Frontal view of a segment of synaptonemal complex. Chromatin loops extend from lateral elements. Sister chromatin loops are represented by thick and thin lines of the same shade of green. At the recombination nodule, DNA loops from two non-sister chromatids are involved in a crossover. Figure 16. Diffuse stage (early diplotene). Homologs desynapse with the disintegration of the SC. However, one or more lateral element proteins may remain in place and other proteins may be added to hold sister chromatids together through the diffuse stage (light gray rods). In the ¢gure, proteins (probably including some from the RN) are associated at the crossover site (gray circle). Figure 17. Late diplotene. (A) Longitudinal view during the transition from the diffuse stage to late diplotene showing new chromatid cores (copper ^ comparable to those in mitotic chromosomes) forming in association with MARs (green dots) at the base of loops comprising each continuous DNA strand. These new cores faithfully follow chromatids though cross overs and thereby form chiasmata. (B) End view of a segment of a diplotene bivalent (not at a chiasma). Sister chromatid cores are close together due to sister chromatid cohesion and lie to one side of the chromatin as was the case for lateral elements in pachytene. Even so, sister loops extend roughly in opposite directions. Also see Figure 20. Figure 18. Diakinesis^metaphase I. Three-dimensional drawing of a bivalent at lower magni¢cation than the diagrams in Figures 13^17. From diakinesis through metaphase I, sister chromatids are held together throughout their length (sister chromatid cohesion) with sister cores to one side of the chromatin. When sister cores actively shorten, sister chromatids coil together. Between chiasmata, coiling can be achieved by including roughly the same number of right- and left-hand twists. Between the end of a bivalent and the ¢rst chiasma, coiling can be all right- or left-handed since the ends can rotate to relieve tension. The bivalent diagrammed in this ¢gure has two chiasmata, and each chromosome has two coiling reversals between the chiasmata. The two sister cores (copper) of each chromosome are indicated by arrows. To the lower right, the relation of chromatin loops and the cores is illustrated. Sister kinetochores (yellow) face the same pole. Also see Figure 22A. Figure 19. Anaphase I. Three dimensional drawing of two homologous anaphase I chromosomes. Sister chromatid cohesion is lost in the arms but maintained at centromeres. As a result, sister chromatid arms swing apart, chiasmata are lost, and homologous chromosomes are pulled to opposite poles by kinetochore microtubules. Also see Figure 22B. 188 S. M. Stack & L. K. Anderson Figure 20. The bar code model for presynaptic alignment of homologous chromosomes is based on two premises: Each chromosome displays a unique sequence of DNA loops in a linear pattern that is analogous to bar codes used for scanning commercial products, and homologous loops of DNA interact weakly to bind them together. (A) Non-homologs (represented by the unlike upper and lower bar codes) have few, if any, like sequences, so they cannot form stable associations when brought into contact. (B) Homologs (represented by the upper and lower identical bar codes) cannot form stable associations unless they are aligned at some point. (C) The bouquet orientation of leptotene and zygotene chromosomes requires movement of chromosomes within the nucleus and encourages alignment of chromosomes starting at their ends on the nuclear envelope (see Zickler & Kleckner 1998 for a review of the bouquet orientation). Indeed, heterozygosity for distal de¢ciencies and asymmetry in isochromosome arms seriously interferes with synapsis, presumably due to dif¢culty in homologous alignment when telomeres are attached to the nuclear envelope (Curtis et al. 1991, Lukaszewski 1997). Once homologous loop interactions begin, homologs (represented by the upper and lower identical bar codes) can zip together by an increasing number of weak, but additive, homologous loop interactions. Chromosome structure through mitosis and meiosis to a lack of MARs/SARs in the insert. In a homozygote for the transgene, all four inserts are nearby and can make contact to form a single FISH signal, probably based on the same weak interactions that lead to presynaptic alignment. In a heterozygote where inserts are on nonhomologs, sister inserts associate to form a single FISH signal, but inserts on non-homologs yield separate signals, i.e. there is no ectopic pairing of inserts on non-homologous chromosomes. This observation could be explained according to the model if sister loop interactions do not leave enough unsaturated sequence to mediate stable ectopic interactions between non-homologs that otherwise would not be attracted to each other. Because chromatin loops are ordinarily contracted, there are always many loops on one homolog that are sterically unavailable for interactions with loops on the other homolog (Figure 13B). In polysomic and polyploid cells there are more than two homologs, so unused DNA loops on an aligned pair of homologs are available to associate with loops on other homologs. The result is that presynaptic alignment is not saturated by associations of chromosome pairs, but can involve simultaneously as many homologs as are present in a cell (e.g. Loidl 1988). Homologous interactions in late leptotene draw homologs into close enough association that transverse ¢lament proteins attached at their C termini to axial cores can interact head to head (N termini to N termini) to form transverse ¢laments that span the central region between the axial cores (Westergaard & von Wettstein 1966, Schmekel et al. 1996, Liu et al. 1996, Dong & Roeder 1999). Once transverse ¢laments are in place, synapsis has occurred, and axial cores are now referred to as lateral elements. Collectively the lateral elements and transverse ¢laments constitute the SC (Figure 14, see Schmekel et al. 1993 for structure of the SC). By drawing axial cores together in this way, there is a tendency for synapsis to proceed in either direction from an initiation site. While synapsis may be completed from a single initiation site (Greenbaum et al. 1986), usually there are multiple sites of synaptic initiation, especially on large chromosomes (e.g. Hasenkampf & Menzel 1985). Generally synapsis is not initiated in heterochromatin (e.g. Stack & Anderson 1986a). 189 Early recombination nodules are ellipsoidal, darkly-stained, proteinaceous bodies that are found in association with axial elements during leptotene and in the central regions of SCs during zygotene (Figure 14; e.g. Stack & Anderson 1986a). Early nodules are often observed between segments of axial cores that approach each other prior to synapsis and at forks where synapsis has just occurred (Albini & Jones 1987, Anderson & Stack 1988, Stack & Roelofs 1996). Although proteins that are capable of mediating DNA^ DNA interactions are components of at least some early nodules (such as Rad51p and Dmc1p; Anderson et al. 1997, Moens et al. 1997a, Tarsounas et al. 1999), it remains unclear whether early nodules are involved in presynaptic alignment or synapsis (von Wettstein et al. 1984, Stack & Anderson 1986b, Zickler & Kleckner 1999). An important feature of this model is that sister DNA loops are contracted on opposite sides of lateral elements. As a result, sister loops are sterically inhibited from interacting with each other during crossing over, gene conversion, or other DNA repair events. In contrast, homologous non-sister DNA loops can come into contact above, below, and possibly through the central region of the SC (Figure 14). This arrangement of sister loops would explain why non-sister chromatids are involved in crossing over in preference to sister chromatids (Haber et al. 1984, Game et al. 1989, Collins & Newton 1994, Schwacha & Kleckner 1994, 1997). Observations of red1 mutants in S. cerevisiae are consistent with this model (Schwacha & Kleckner 1997). The RED1 gene encodes a lateral element protein. Mutants of RED1 do not form lateral elements, and they do not have an interhomolog bias in crossing over. Schizosaccharomyces pombe and Aspergillus nidulans are also relevant to this discussion because both go through meiosis without forming SCs and yet both have high levels of crossing over without crossover interference (Olson et al. 1978, Egel-Mitani et al. 1982). Because axial elements appear to be formed in S. pombe (Olson et al. 1978, BÌhler et al. 1993), while axial elements seem not to be formed in A. nidulans (Egel-Mitani et al. 1982), this model predicts that S. pombe will show a bias against crossing over between sister chromatids, while A. nidulans will not. However, because Scherthan et al. (1994) report that S. 190 pombe is unusual in having no chromatin condensation at prophase I, it is possible that sister loops can make contact during prophase I to permit a bias towards sister strand crossing over in this species as well. In regard to recombination nodules that are probably involved in the molecular events associated with gene conversion and crossing over (Carpenter 1975 and subsequently many additional references), their location on one side or the other of the central region of an SC would permit a recombination nodule to be in contact with homologous non-sister DNA loops much more easily than with sister DNA loops (Figure 14). Again this would bias crossovers toward non-sister chromatids. However, plant and animal mutants that do not form lateral elements or SCs have essentially no crossing over between homologs (Moses 1968), and fungal mutants that do not form lateral elements also have reduced or eliminated crossing over (e.g. Smith & Roeder 1997, van Heemst & Heyting 2000). Because of this, one must conclude that lateral elements and SCs play a more active role in crossing over than simply suppressing crossing over between sister chromatids. Most likely this involves association of recombination nodules with axial/lateral elements (Figures 14 & 15; e.g. Stack & Anderson 1986a, 1986b, Storlazzi et al. 1996). SCs are sometimes observed to be twisted (Zickler & Kleckner 1999). While a variety of explanations have been offered to account for twisting of whole SCs, a simple explanation is based on active shortening (contraction) of the lateral elements that are attached to relatively incompressible chromatin on one side and rigid transverse ¢laments on the other side. If transverse ¢laments can swivel or bend to some degree at their attachment sites to lateral elements, contraction of lateral elements will cause them to spiral around a longitudinal axis with the chromatin to the outside (demonstrated in a rope and elastic model but not illustrated). Breakdown of SCs marks the end of pachytene and the beginning of diplotene. In many organisms, this point is referred to as the diffuse stage because nuclei take on an interphase-like appearance (Figure 16; e.g. Moses 1968, von Wettstein et al. 1984, see Wilson 1928 and Kläs̄terskä 1976, 1977 for reviews of the diffuse S. M. Stack & L. K. Anderson stage). Breakdown of SCs is a necessary prerequisite for reformation of chromatid cores that re£ect the results of crossing over, i.e. chiasmata (e.g. Rufas et al. 1982, Stack 1991). While some SC proteins are lost from chromosomes at this time, a few lateral element proteins remain with the chromosomes, particularly at the centromeres (e.g. Moens et al. 1987, Moens and Spyropoulos 1995, Molnar et al. 1995, Klein et al. 1999, Zickler and Kleckner 1999). Some of these lateral element proteins may be incorporated into newly formed chromosome cores (e.g. Moens & Earnshaw 1989, Fedotova et al, 1989, van Heemst & Heyting 2000). Considering the position of the diffuse stage in the meiotic cell cycle, this interphase-like stage is probably equivalent to G2 in a mitotic cell cycle. Indeed, since G2 is de¢ned as that part of interphase after S phase and before chromosomes condense at prophase (Howard & Pelc 1953 and see below), it is attractive to interpret leptotene, zygotene, pachytene, and the diffuse stage collectively as a long G2 phase during which structures (axial cores, SCs, early and late recombination nodules) that are needed for crossing over and gene conversion between homologous nonsister chromatids form, carry out their functions, and break down (also see Beerman 1963, van Heemst & Heyting 2000). There is a great deal of transcription during the diffuse stage (Kläs̄terskä 1977), and this is the stage when lampbrush chromosomes appear in primary oocytes. Indeed, interpreting the diffuse stage as the end of G2 suggests that lampbrush chromosomes represent highly extended and extensively transcribed late interphase chromosomes. For the chromosome model presented here, it is of interest that sister loops probably extend to opposite sides of lampbrush chromosome axes (Figure 21 and see other illustrations in Callan & Lloyd 1963 and Callan 1986) just as sister loops are hypothesized to emerge from opposite sides of mitotic G2 nuclear matrix strands, axial elements, and lateral elements. After the diffuse stage, long diplotene chromosomes become visible (Figure 17). As chromosomes shorten in late diplotene and diakinesis, a pair of closely associated sister cores that can be stained with silver becomes visible in each chromosome (Rufas et al. 1982, 1987, Stack 1991, Gimënez-Abiän et al. 1997). These paired cores Chromosome structure through mitosis and meiosis 191 Figure 21. Camera lucida drawing of a lampbrush chromosome from a crested newt (Triturus cristatus (Laurenti)) primary oocyte. Note that sister loops of chromatin extend in opposite directions from the axes. [Callan, HG and Lloyd, L (1963), Lampbrush chromosomes of crested newts Triturus cristatus (Laurenti). Roy Soc Lond Phil Trans Ser B 243: 135^219, Figure 9a; reproduced with permission.] are comparable to those in paired sister chromatids of mitotic prophase chromosomes (Gimënez-Abiän et al. 1995), except diplotene cores trade partners at chiasmata (compare Figures 9 & 17). While it is unclear how newly formed chromosome cores accurately track DNA through chiasmata, the problem may be no different and the mechanism the same as when newly formed sister cores track sister DNA double helices in G2-prophase of mitosis. During diplotene^metaphase I, sister chromatid cohesion maintains chiasmata and keep homologs together (Darlington 1932, Maguire 1974). In grasshoppers, sister cores are close together in the center of the chromatin mass and, as a result, there is no coiling (Rufas et al. 1987). In Lilium longi£orum (lily), sister chromatids coil together during diakinesis-metaphase I (Stack 1991). These pairs of sister cores are close but separate and lie to one side of the chromatin of the sister chromatids (Figure 17B). In this lateral position, active contraction of the sister cores during diplotene^diakinesis results in coiling, just as in mitosis (Figures 18 & 22A). Because there are two close, parallel sister cores (rather than the separate single sister cores observed during mitosis), the major coils of lily chromosomes at diakinesis have larger gyres than lily mitotic chromosomes (Stack 1991). Distal to chiasmata, the strain of coiling is relieved by twisting sister chromatids around each other in the same direction as the coiling. However, strain caused by chromosome coiling between chiasmata cannot be relieved like this. Instead, there are reversals of the direction of coiling to yield little or no net coiling or twisting between chiasmata. Active contraction of cores and coiling of chromosomes during diplotene^ diakinesis, causes them to shorten and thicken. This puts tension on chiasmata and causes chromosomes to bulge out between chiasmata, a phenomenon sometimes referred to as repulsion (Figures 18 & 22A; Ostergren 1943). It should be mentioned that certain plants (such as Tradescantia and lily) have very large meiotic chromosomes that have sometimes been reported to consist of large (major) coils of a smaller (minor) coiled strand (e.g. Kuwada 1938, Manton 1950, Taylor 1958, Stack 1991). If, indeed, two levels of coiling can coexist in the same chromosome, this is not readily explained with a single contractile core in each chromatid. The chromosome model proposed here provides an explanation for how the two pairs of sister kinetochores in a metaphase I bivalent are oriented to opposite poles (amphitelic orientation). The geometry of a coiled bivalent dictates that one pair of sister kinetochores is located to one side of the chromatin mass while the other pair is located to the other side. In addition, the side-by-side arrangement of sister chromatids during diakinesis^metaphase I positions both sister kinetochores on one side of the chromosome so microtubules from one pole attach to both of them (syntelic orientation), while the other two sister kinetochores on the homologous chromosome attach to microtubules from the opposite pole (syntelic orientation) to establish amphitelic orientation for the whole bivalent (Figures 18 & 22A). At anaphase I, sister chromatid binding is lost in the arms, but not at centromeres (Figures 19 & 22B; e.g. Moens & Spyropoulos 1995, Sekelsky 192 S. M. Stack & L. K. Anderson Figure 22. Rope and elastic models of meiotic chromosomes. The chromatin of each chromatid is represented by a segment of white rope that is 53 cm long in all models. The chromatid core is represented by a black elastic band that was sewn to the white rope. Kinetochores (K) are indicated by felt patches. In these models, 1 cm represents 1 mm, i.e. the ropes are 10 000 : 1 scale models of chromosomes. (A) Diakinesis^metaphase I chromosome. To prepare the model, a 53-cm length of white rope was sewn to a length of elastic maximally stretched to 53 cm. This represents one chromatid. This was repeated three more times for a total of four ropes four chromatids. While still maximally stretched, the proximal two-thirds of two lengths of rope/elastic were sewn together side-by-side to represent one homolog, and the proximal two-thirds of the other two lengths of rope/elastic were similarly sewn together to represent the other homolog. The ropes were not sewn together elastic band to elastic band because the cores look nearby but separate in lily prometaphase I chromosomes (Stack 1991). For a distal chiasma, one rope end ( chromatid) from one rope pair ( homolog) was sewn in the same orientation to another rope end ( chromatid) from the other rope pair ( homolog). This process was repeated for the other rope ends. The pairs of rope ends distal to a partner trade represent pairs of sister chromatids held together by sister chromatid cohesion. The same sewing was repeated to represent another distal chiasma at the other end of the `bivalent'. Then, the tension on all four ropes was released to permit coiling. Reversals of coiling occur between chiasmata because tension cannot be relieved by rotating the ends of the chromosomes. Two pairs of sister kinetochores (K) face in opposite directions. Sister cores (double arrows) to the inside of the coils. Also see Figure 18. (B) Separating anaphase I chromosomes. In this model, each of four 53-cm lengths of white rope was sewn to a 53 cm length of maximally stretched elastic band. While still maximally stretched, two of the ropes were sewn together side-by-side for a short distance at the centromeres to represent sister chromatid cohesion that is still present at this location. The ropes were sewn together side-by-side as a continuation of their relationship from metaphase I. When tension on the ropes was released, the ropes coiled along their length with the sister kinetochores facing outward in the same direction. A second model of an anaphase I chromosome was prepared in the same way using the other two lengths of rope/elastic. Then the two models were arranged as they would look separating at anaphase I. Also see Figure 19. Chromosome structure through mitosis and meiosis & Hawley 1995, Klein et al. 1999). With the loss of sister chromatid cohesion, each arm coils independently. However, because the sister centromeres are still held together in a side-by-side orientation, the coiled arms swing apart in a manner consistent with Ostergren's (1943) model of the response of two elastic bodies bound together at a point. Loss of sister chromatid cohesion along the arms eliminates chiasmata, and homologous chromosomes are pulled to opposite poles by tension on kinetochore microtubules. Telophase I and a modi¢ed interphase called interkinesis occur between meiotic divisions. The chromosomes decondense, uncoil, and lengthen, but there is no DNA synthesis and no reorganization of chromosome structure. Chromosomes that recondense at prophase II are little altered from anaphase I. Indeed, many animal eggs largely or completely skip telophase I and interkinesis to move directly into prophase II (Sharp 1926). In either case, sister kinetochores still lie to one side of metaphase II chromosomes (e.g. Gimënez-Abiän et al. 1997) but anaphase II cannot begin until sister kinetochores are under tension due to establishing amphitelic orientation on the spindle (Nicklas et al. 1995). The second meiotic division is otherwise a typical mitotic division of a haploid cell (Figures 7^11 & 12B^E). Conclusion We propose three important additional features to the chromosome scaffold model: (1) DNA loops extend from only one side of a chromatid core; (2) chromatid cores are actively contractile; and (3) after DNA replication, sister DNA loops extend in opposite directions from sister cores. Together, these proposed features provide a mechanical explanation for chromatid coiling and a steric explanation for the preferential involvement of non-sister chromatids in crossing over. Based on these proposals, we make speci¢c testable predictions about chromosome structure. 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