InteractiveFly: GeneBrief

orientation disrupter: Biological Overview | References

During meiosis, cohesion between sister chromatids is required for normal levels of homologous recombination, maintenance of chiasmata and accurate chromosome segregation during both divisions. ORD activity is essential for the crucial decision each chromatid must make after the induction of DSBs (double strand breaks) – namely whether the broken chromatid will choose its sister or its homologue for repair. Strand invasion and crossovers are biased towards the homologue during meiosis, resulting in stable chiasmata that keep homologous chromosomes physically associated until anaphase I. In Drosophila, null mutations in the ord gene abolish meiotic cohesion, although how ORD protein promotes cohesion has remained elusive. This study shows that SMC (structural maintenance of chromosome) subunits of the cohesin complex (see A Ring for Holding Sister Chromatids Together?) colocalize with ORD at centromeres of ovarian germ-line cells. In addition, cohesin SMCs and ORD are visible along the length of meiotic chromosomes during pachytene and remain associated with chromosome cores following DNase I digestion. In flies lacking ORD activity, cohesin SMCs fail to accumulate at oocyte centromeres. Although SMC1 and SMC3 localization along chromosome cores appears normal during early pachytene in ord mutant oocytes, the cores disassemble as meiosis progresses. These data suggest that cohesin loading and/or accumulation at centromeres versus arms is under differential control during Drosophila meiosis. The experiments also reveal that the α-kleisin C(2)M is required for the assembly of chromosome cores during pachytene but is not involved in recruitment of cohesin SMCs to the centromeres. A model is presented for how chromosome cores are assembled during Drosophila meiosis and the role of ORD in meiotic cohesion, chromosome core maintenance and homologous recombination (Khetani, 2007).

Accurate segregation of chromosomes during meiosis relies on a number of dynamic changes in chromosome morphology that take place within the context of sister-chromatid cohesion. Meiotic cohesion is not only required for the correct segregation of sisters during the second meiotic division, but also ensures that recombinant homologous chromosomes remain physically associated until anaphase I. In addition, arm and centromeric cohesion must be regulated differently during meiosis. When the release of arm cohesion during meiosis I allows the segregation of homologues, centromeric cohesion must be protected and remain intact until anaphase II when sisters segregate to opposite poles (Khetani, 2007).

Cohesion between meiotic sister chromatids plays an essential role in assembly of the synaptonemal complex (SC), a tripartite proteinaceous structure that forms between homologous chromosomes during prophase I. During early prophase I, each pair of sister chromatids undergoes shortening along their longitudinal axes, resulting in the formation of 'chromosome cores' upon which the axial/lateral elements (AEs/LEs) of the SC assemble. During pachytene, SC central element proteins join each set of homologous AEs/LEs along their entire length resulting in synapsis of homologues. In many species (yeast, mice, Arabidopsis), meiotic double-strand breaks (DSBs) are essential for homologue synapsis; however, chromosome core formation (axial shortening) does not depend on DSBs. In addition, mutants that lack AE/LE components can still build chromosome cores (Khetani, 2007).

Crossovers between homologous chromosomes, in conjunction with sister chromatid cohesion, are essential for correct chromosome segregation during meiosis I. In most organisms, recombination between homologues takes place in the context of the SC. Although EM studies indicate that the ultrastructure of the SC is highly conserved, SC components in different organisms show surprisingly little sequence homology (Khetani, 2007).

During both mitosis and meiosis, sister-chromatid cohesion is mediated by an evolutionarily conserved protein complex called cohesin that contains two SMC and two non-SMC subunits (Lee, 2001; Petronczki, 2003). The α-kleisin subunit (Scc1/Mcd1/Rad21) bridges the two head domains of the SMC1-SMC3 dimer and thereby forms a ring that entraps DNA (Nasmyth, 2002; Shintomi, 2007). Several meiosis-specific cohesin subunits have been identified, including the α-kleisin Rec8, which has been shown to be crucial for meiotic cohesion and SC formation in all organisms examined (Khetani, 2007).

In Drosophila, four cohesin subunits have been uncovered through sequence analysis and the localization and function of mitotic cohesin has been examined in Drosophila embryos and tissue culture cells. However, little is known about the localization and dynamics of the cohesin complex during Drosophila meiosis (Khetani, 2007).

Drosophila oogenesis is an excellent system to study meiosis, as each Drosophila ovary is composed of approximately 10-30 ovarioles that contain a linear array of oocytes at progressive developmental stages from mitotic germ-line stem cells to metaphase-I-arrested oocytes. Meiosis initiates in the germarium, the most anterior structure of each ovariole. Germ-line stem cells in region 1 of the germarium undergo four rounds of synchronous mitotic divisions resulting in 16 interconnected cells that comprise a 'cyst'. As cysts mature, they move toward the posterior end of the ovariole. All germ cells within a 16-cell cyst undergo pre-meiotic S phase synchronously and prophase I of meiosis initiates in germarial region 2A where up to four cells per cyst initiate SC assembly. In addition, meiotic DSBs are induced in region 2A, but unlike several other organisms, synapsis in Drosophila does not depend on DSBs. As each cyst moves through the germarium, the SC breaks down in all but one nucleus so that, by region 3, full-length SC is restricted to the oocyte, which lies at the posterior end of the rounded cyst. As cysts continue to grow and mature, they leave the germarium and move into the 'vitellarium'. The oocyte remains in pachytene with full-length SC until vitellarial stage 6; however, the remaining 15 cells within each cyst adopt a nurse cell fate and enter an endo cell cycle, during which multiple rounds of S phase in the absence of intervening M phase results in polyploid cells (Khetani, 2007).

Meiotic cohesion in Drosophila depends on the novel protein, Orientation Disruptor (ORD) (Bickel, 1996; Bickel, 1997; Mason, 1976; Miyazaki, 1992). In mutants lacking ORD function, sister-chromatids segregate randomly through both meiotic divisions, consistent with complete absence of meiotic cohesion (Bickel, 1997). In addition, homologous recombination is severely reduced in ord^null females and SC assembly and maintenance are disrupted (Bickel, 1997; Webber, 2004). Immunolocalization studies have demonstrated that ORD is enriched at the centromeres of meiotic chromosomes in both males and females (Balicky, 2002; Webber, 2004). In addition, ORD localization along the arms of female meiotic chromosomes coincides with that of the SC protein, C(3)G (Webber, 2004; Khetani, 2007 and references therein).

This study investigated the localization and dynamics of two cohesin subunits (dSMC1 and Cap/dSMC3) during early prophase I in Drosophila oogenesis. SMC1 and SMC3 localize along the arms and are enriched at the centromeres of all 16 cells within each germ-line cyst. In nuclei that build SC, cohesin subunits coalesce into chromosome cores that provide the scaffold for SC assembly. Formation of chromosome cores depends on the α-kleisin C(2)M, and the cohesion protein ORD is essential for cohesin loading at centromeres and for maintenance of chromosome cores. These data support the argument that during meiosis, the establishment of centromeric cohesion is regulated differently than on the arms. Moreover, these results provide insight into the interconnected roles of meiotic cohesion, chromosome cores and homologous recombination (Khetani, 2007).

Accumulation of cohesin SMCs on chromosomes of pre-meiotic cells: To analyze the behavior of cohesin subunits during meiotic progression in the Drosophila ovary, antibodies were generated against Drosophila SMC1 and SMC3 peptides. Following affinity-purification, SMC1 and SMC3 antibodies each recognize a single predominant band at the predicted molecular mass in embryo extracts and a doublet/triplet in ovary extracts. In addition, when germ-line clones are generated that are homozygous for the smc1 excision allele, smc1^exc46, no SMC1 signal above background is observed. Because affinity-purified SMC1 and SMC3 antibodies display very similar staining patterns in Drosophila ovaries, they were combined for most of the experiments described below to maximize signal intensity (here referred to as SMC1/3 or SMC) (Khetani, 2007).

Fixation and staining of intact ovarioles (whole-mount preparations) revealed several distinct cohesin SMC staining patterns within the germaria of wild-type females; multiple regions of each germarium contained bright foci, as well as diffuse staining and nuclei with a thread-like SMC signal. Although no cohesin SMC staining was detected in germarial stem cells and early cystoblasts in region 1, bright foci as well as diffuse SMC localization are visible in the nuclei of germ-line cysts within region 1. The bright SMC foci correspond to centromeres as confirmed by co-staining with CID, a centromere-specific histone H3 variant. The diffuse staining in pre-meiotic cells most probably corresponds to cohesin localization along chromosome arms. This same localization pattern has been observed (Webber, 2004) for the cohesion protein ORD in germ-line mitotic cysts (Khetani, 2007).

Thread-like cohesin SMC signal coincides with the SC in pachytene nuclei: As 16-cell cysts enter region 2A of the germarium, up to four nuclei in each cyst begin to assemble a SC and in these cells, SMC1/3 signal becomes visible as thread-like staining that coincides with the SC marker C(3)G. During the maturation of cysts and their progression through the germarium, thread-like SMC1/3 staining mimics that of the SC. As cysts move through the germarium, continuous linear SMC1/3 staining is visible in the two nuclei that contain full-length SC (pro-oocytes) but the SMC1/3 signal appears fragmented in the other C(3)G containing nuclei that will adopt a nurse cell fate (pro-nurse cells). Oocyte determination is complete by region 3 and, at this stage, the continuous thread-like SMC1/3 staining is restricted to the oocyte (Khetani, 2007).

The oocyte nucleus will remain in pachytene for several hours as it progresses through the vitellarium. Electron microscopy has shown that that full-length tripartite SC is present as late as stage 6 of vitellarial development and these data have been supported by persistence of continuous threads of C(3)G immunostaining until the same stage (Page and Hawley, 2001). In vitellarial stages 2 to 6, thread-like SMC1/3 staining is observed in whole-mount preparations that is coincident with C(3)G signal. However, similar to C(3)G staining, the thread-like signal becomes weaker in these later stages and is accompanied by increased diffuse nuclear staining (Khetani, 2007).

Cohesin SMCs and ORD are present along chromosome cores during pachytene: Thread-like signals for cohesin SMCs as well as the cohesion protein ORD are restricted to germ-line cells that form SC (Webber, 2004). One possibility is that, together, these proteins contribute to the proteinaceous 'chromosome core' that has been proposed to serve as a scaffold for SC formation (Revenkova, 2006; Stack, 2001). If the cohesin complex and ORD are indeed part of the chromosome core, they should persist in the absence of DNA loops in SC-containing nuclei. To test this hypothesis, chromosome spreads of germarial cells were prepared to visualize proteins bound to meiotic chromosomes. In this procedure, soluble components are washed away, leaving only chromosomes and their associated proteins attached to the slide. DNase I treatment of chromosome spread slides resulted in loss of histone and DAPI staining, confirming that DNA loops had been digested. However, the thread-like SMC1/3 and ORD staining persisted in the absence of DNA loops, consistent with the model that ORD and the cohesin complex are components of the cores of meiotic chromosomes in SC-forming nuclei (Khetani, 2007).

Chromosome spread experiments also revealed that cohesin SMCs are associated with chromosome arms in all 16 nuclei of each germ-line cyst. However, in nuclei that do not build a SC, the SMC localization pattern is diffuse rather than thread-like. In addition, it was observed that during SC disassembly in non-oocyte nuclei, cohesin SMCs remain associated with chromosome arms and their staining pattern is indistinguishable from other pro-nurse cells. Association of ORD with chromosome arms in a pattern similar to SMC1/3 has been described previously (Webber, 2004). Soluble nuclear proteins are removed during the spread preparation. When transgenic flies expressing GFP-nls were used to generate spreads, diffuse SMC1 staining was visible in several nuclei but no corresponding GFP signal was detected. Therefore, it is concluded that the diffuse SMC1/3 staining observed in chromosome spreads represents cohesin SMCs stably associated with the chromatin. These data support the model that cohesin SMCs and ORD associate with the arms of all germ-line chromosomes and in cells that build SC, cohesion proteins coalesce into continuous threads that represent chromosome cores. Interestingly, diffuse SMC staining is often not visible in spread preparations of SC containing nuclei, suggesting that most or all of the cohesin complex in these cells is located along the cores, not the loops of meiotic chromosomes (Khetani, 2007).

Enrichment of cohesin SMCs at the centromeres of meiotic chromosomes: In addition to the thread-like staining pattern in pachytene cells, cohesin SMCs are enriched at the centromeres of wild-type meiotic chromosomes as confirmed by colocalization of the SMC1/3 foci with CID, the centromere specific histone H3 variant. The centromeres of Drosophila chromosomes are usually clustered together into a single chromocenter and each bright focus of SMC1/3 and ORD staining that is visible in the nuclei of whole-mount preparations corresponds to the chromocenter. In chromosome spreads, the chromocenter is frequently observed split into two or more regions. Interestingly, the increased resolution afforded by spread preparations indicates that the bright SMC1/3 signal at centromeres often extends beyond the area of CID staining. Cohesin SMCs exhibit the same extensive centromeric localization pattern as ORD (Webber, 2004), consistent with enrichment of these cohesion proteins within pericentric as well as centromeric heterochromatin. Robust SMC1/3 and ORD signals in the vicinity of the centromere are not restricted to nuclei that build SC. Instead, centromeric enrichment of these proteins is visible in all 16 cells of germarial cysts (Webber, 2004) whether they adopt a nurse cell or oocyte fate (Khetani, 2007).

As egg chambers progress into the vitellarium, the SMC1/3 signal associated with the chromocenter in nurse cells begins to assume a very distinctive pattern that resembles a cluster of finger-like projections. Interestingly, the onset of this staining pattern coincides with the beginning of the endo-reduplication cell cycle in nurse cells, during which DNA replication occurs repeatedly in the absence of cell division. These SMC1/3 finger-like projections are observed during early vitellarial stages when the polyploid nurse cell chromosomes exhibit polyteny, the precise alignment of multiple copies of sister chromatids. Unlike polytene chromosomes in the Drosophila salivary gland, nurse cell polytene chromosomes are short-lived. Around vitellarial stage 4, nurse cell chromosomes undergo a dramatic morphological change and no longer exhibit polyteny. Although the SMC1/3 signal remains enriched at the pericentric heterochromatin as nurse cell chromosomes transition out of polyteny, the pattern becomes more diffuse and less structured in these later stages (Khetani, 2007).

ORD is required for centromeric localization of cohesin SMCs during meiosis: ORD protein is necessary for both arm and centromeric cohesion during Drosophila meiosis (Bickel, 1996; Bickel, 1997; Mason, 1976). In mutant flies lacking ORD activity, chromosomes segregate randomly through both meiotic divisions, indicating that cohesion is completely absent (Bickel, 1996; Bickel, 1997; Mason, 1976). The localization pattern of ORD protein during early oogenesis (Webber, 2004) closely mimics that of the cohesin SMC proteins. One possibility is that ORD controls the localization and/or function of the cohesin complex during meiosis. To study the localization dynamics of the cohesin complex in the absence of ORD, ovaries from ord⁵/Df (ord^null) females were examined. The ord⁵ mutation results in premature truncation of the ORD open reading frame and genetically behaves like a null-allele (Bickel, 1996; Bickel, 1997; Khetani, 2007).

When whole-mount ord^null germaria are stained with SMC1 and SMC3 antibodies, bright centromeric foci are conspicuously absent throughout the germaria even though continuous thread-like staining is visible in region 2A. Within the germarium, SMC1/3 foci are undetectable in cells that form SC as well as the remaining cells of each cyst. ord^null oocytes also lack SMC1/3 centromeric foci after they exit the germarium. These data suggest that ORD is essential for normal accumulation of cohesin at oocyte centromeres and are consistent with the chromosome segregation defects observed in mutant flies (Bickel, 1996; Bickel, 1997; Miyazaki, 1992). Absence of cohesin SMC localization at centromeres in the ord mutant was confirmed when chromosome spreads were immunostained for SMC1/3 and the centromere marker CID. Cohesin SMCs do not colocalize with CID foci in ord^null germaria; the CID signal corresponds to gaps in the thread-like SMC1/3 signal. These data suggest that ORD activity is required for loading and/or accumulation of centromeric cohesin during female meiosis (Khetani, 2007).

Interestingly, although centromeric SMC1/3 staining is never visible in oocytes of ord^null flies, a distinct centromeric staining pattern becomes detectable in nurse cells as cysts progress into the vitellarium. Even in the absence of ORD, finger-like projections of SMC1/3 staining were observed in the vicinity of nurse cell centromeres in ord^null mutant egg chambers by vitellarial stage 3, presumably when polytene chromosomes are present. Like wild type, this SMC1/3 staining becomes diffuse at later stages when polyteny is absent. These data argue that loading of cohesin subunits onto centromeres is controlled differently in oocytes and nurse cells, and once germ-line cells adopt a nurse cell fate, accumulation of cohesin at centromeres is no longer dependent on ORD function (Khetani, 2007).

ORD is necessary for maintenance of chromosome cores during early meiosis: Despite the centromeric defects that were observe in ord^null germaria, thread-like SMC1 and SMC3 staining along chromosome cores appear relatively normal during early pachytene even in the absence of ORD activity. In early region 2A (the anterior portion of region 2A), long continuous threads of SMC1 and SMC3 are visible in ord^null germaria, although SMC staining along cores is weaker than in wild type. However, both the intensity and integrity of cohesin thread-like staining deteriorates progressively as cysts mature and travel through the germarium. A gradual loss of thread-like C(3)G staining as cysts mature has also been observed (Webber, 2004) in ord^null germaria (Khetani, 2007).

To characterize the progressive deterioration of SMC1 and SMC3 thread-like staining in ord^null germaria, the integrity and intensity of the threads were scored in different regions of wild-type and mutant germaria. Careful analysis of the defects in several mutant germaria indicated that, by late region 2A (the posterior portion of region 2A), the intensity of the SMC1 and SMC3 thread-like staining was significantly reduced and fragmented threads were visible in a number of cells. For example, in late region 2A, a pronounced reduction in SMC1 signal intensity was observed in 43% of ord^null cysts but only 3% of wild-type cysts. Similarly, fragmented SMC1 threads were observed in 15% of late region 2A mutant cysts, but no fragmentation was visible at this stage in wild type. In older mutant cysts, loss of the thread-like SMC1 and SMC3 staining became more prominent. By region 3, no mutant oocyte nucleus exhibited robust continuous thread-like SMC1 or SMC3 staining. At this stage, 45% of ord^null oocyte nuclei contained no visible SMC1 staining and 53% contained severely fragmented threads. SMC3 signal was undetectable in approximately 52% of region 3 ord^null oocyte nuclei and about 30% had short dim fragments. Interestingly, the anti-SMC1 and anti-SMC3 antibodies appear to have different affinities for their respective antigens in wild-type nuclei; SMC1 signal along chromosome cores was consistently more robust than that for SMC3. This difference may reflect variation in epitope accessibility for the two proteins and is most likely the cause for quantitative differences in the defects observed for SMC1 and SMC3 in mutant germaria. However, deterioration of the thread-like signal followed the same trend for both proteins and reinforces the conclusion that ORD activity is required to maintain chromosome cores during early pachytene. Notably, these defects first become manifest after the onset of homologous recombination, namely the induction of DSBs in region 2A (Khetani, 2007).

ORD is not required for stable association of cohesin SMCs with chromosome arms during pachytene: The loss of thread-like staining in whole-mount preparations of ord^null germaria initially suggested that cohesin dissociates from chromosome arms during pachytene in the absence of ORD activity, consistent with the essential role of ORD in arm cohesion. However, it was reasoned that it was also possible that cohesin SMCs might remain associated with chromatid arms in the absence of ORD, but loss of thread-like staining might occur because the longitudinal compaction of meiotic chromosome cores depends on ORD function. If cohesin SMCs remain associated with chromosome arms during pachytene in ord^null females but chromosome cores are unstable, the thread-like SMC1/3 signal would disappear. However, it is difficult to detect diffuse localization of cohesin SMCs along chromosome arms in whole-mount preparations. Therefore, chromosome spreads were prepared from ord^null germaria and immunostained for SMC1/3 and C(3)G proteins. Because in spread preparations, the temporal arrangement of individual cysts within each germaria is not maintained, semi-intact cysts were sought that contained a maximum of one or two nuclei with C(3)G staining, reasoning that these most probably represent region 2B cysts. At this stage in ord^null germaria, C(3)G thread-like signal has begun to fragment (Webber, 2004). Diffuse SMC1/3 staining is readily evident in nuclei that also contain fragmented C(3)G and SMC1/3 threads. Moreover, the intensity of diffuse SMC1/3 signal in these nuclei is very similar to that of adjacent pro-nurse cell nuclei. Because soluble nuclear protein is removed during the spread preparation, the diffuse SMC1/3 signal that was observed represents cohesin subunits that are associated with the chromatin but not organized into chromosome cores. These data argue that, in the absence of ORD activity, chromosome cores disassemble but cohesin SMCs remain associated with chromosome arms (Khetani, 2007).

Temporal relationship between SMC1/3 and C(3)G defects in ord^null oocytes: The progressive deterioration of chromosome cores in ord^null germaria is reminiscent of the fragmentation and loss of thread-like staining observed by Webber (Webber, 2004) for the SC central element component, C(3)G. However, a careful comparison of the quantitative analyses of cohesin SMC and C(3)G localization defects (Webber, 2004) indicates that the onset of SMC1/3 localization defects appear to precede those for C(3)G. This is not completely unexpected given that chromosome cores have been proposed to serve as the scaffold upon which SC axial/lateral and central element components can assemble. If disruption of chromosome cores in ord^null oocytes causes a subsequent loss of the central element between homologues, defects in the C(3)G staining pattern should closely follow disintegration of SMC1/3 threads. To test this hypothesis, SMC1/3 and C(3)G defects were examined simultaneously in individual nuclei at different stages in whole-mount preparations of intact ord^null germaria (Khetani, 2007).

Continuous thread-like staining is evident for both C(3)G and SMC1/3 in early region 2A nuclei of ord^null germaria with extensive overlap between the two signals. However, by late region 2A, the SMC1/3 signal appears more fragmented than the C(3)G signal, with fewer SMC1/3 threads and more punctate staining. This difference is most obvious in region 3, where intact threads of C(3)G staining are still evident but the SMC1/3 pattern consists primarily of puncta. These data support the hypothesis that premature breakdown of chromosome cores induces the defects in C(3)G staining that were observed in ord germaria. However, the residual C(3)G threads that remain when SMC1/3 thread-like staining disappears raises the intriguing possibility that aligned C(3)G proteins might form polymers that remain transiently stable, even if they are no longer associated with chromosome cores (Khetani, 2007).

It was also asked whether C(3)G is required to maintain chromosome core integrity; thread-like SMC1/3 staining is still visible in c(3)G⁶⁸/Df mutant germaria in which the SC fails to form. These data indicate that intact SC is not necessary for chromosome core formation or maintenance. Interestingly, SMC1/3 threads appear more numerous in c(3)G mutant nuclei than in wild type, consistent with the inability of homologues to synapse in the absence of C(3)G. In the absence of synapsis, the homologous cores would not be intimately associated and cohesin staining would be visible along individual chromosome cores. A similar staining pattern has been reported for the putative lateral element component C(2)M in c(3)G mutants (Manheim, 2003) providing further support for this model (Khetani, 2007).

C(2)M is required for chromosome core formation during pachytene: To explore the mechanistic interplay between cohesion proteins (ORD, cohesin SMCs), and an α-kleisin involved in SC assembly (C(2)M), the localization of SMC1/3 and GFP-ORD were examined in females homozygous for the c(2)M-null allele, c(2)M^EP[2115] (Manheim, 2003). To observe ORD localization, a c(2)M^EP[2115] stock homozygous for a functional GFP-ORD transgene (Balicky, 2002) was generated and it was confirmed that X-chromosome meiotic nondisjunction in c(2)M^EP[2115];P{GFP-ORD} females (24.44%, n=753) was similar to that previously reported for c(2)M^EP[2115] homozygotes (29.3%) (Manheim, 2003; Khetani, 2007).

At first glance, the staining pattern for SMC1/3 and GFP-ORD in c(2)M^[EP2115] germaria appeared very similar to that previously observed for C(3)G in this mutant (Manheim, 2003). Thread-like SMC1/3 staining is completely absent in the germaria of whole-mount ovaries. Instead, SMC1/3 staining is restricted to patches and foci (Khetani, 2007).

In c(2)M^[EP2115] females, the chromosome segregation defects are severe in meiosis I, but negligible during meiosis II (Manheim, 2003). This suggests that centromeric cohesion in these mutants is intact. To test whether the patches of SMC1/3 and GFP-ORD staining correspond to centromeres, c(2)M^[EP2115] ovaries were co-immunostained with anti-CID antibodies. In these whole-mount preparations, CID foci largely coincide with ORD and SMC1/3 patches in pro-oocyte and oocyte nuclei in all regions of c(2)M-mutant germaria. These data argue that neither loading nor maintenance of cohesin SMCs at centromeres depends on the activity of C(2)M protein, consistent with low levels of meiosis II segregation defects in c(2)M-mutant females (Manheim, 2003; Khetani, 2007).

Absence of thread-like SMC1/3 signal in whole-mount preparations of c(2)M-mutant germaria raises the possibility that C(2)M activity is required for loading cohesin subunits onto chromosome arms. Alternatively, cohesin subunits may localize normally to the arms, but fail to coalesce into chromosome cores in the absence of C(2)M. In this case, diffuse chromatin-bound cohesin signal would probably go undetected in whole-mount ovary preparations. To address this possibility, chromosome spreads prepared from c(2)M^EP[2115];P{GFP-ORD} germaria were examined. In the c(2)M mutant, each nucleus contained one to four centromeric foci in which SMC1/3 and GFP-ORD always colocalized. These foci also coincided with C(3)G foci in the subset of cells that contained C(3)G signal. This staining pattern is consistent with that observed in whole-mount preparations of c(2)M germaria. However, in the chromosome spreads, diffuse chromosomal ORD and SMC1/3 staining also was observed in all mutant c(2)M pro-oocytes and pro-nurse cells. Moreover, the intensity of diffuse SMC1/3 and ORD signal is comparable in nuclei with and without C(3)G patches/foci (pro-oocytes and pro-nurse cells, respectively) (Khetani, 2007).

The data for chromosome spread localization indicate that in the absence of C(2)M, ORD and cohesin SMCs are loaded and maintained on both the arms and centromeres of meiotic chromosomes. However, absence of thread-like ORD and SMC1/3 staining argues that assembly of chromosome cores requires C(2)M activity. Because stable chromosome cores and lateral elements are a prerequisite for SC formation, their absence most probably explains the lack of thread-like C(3)G signal in c(2)M mutant germaria (Khetani, 2007).

Summary: This study has describe temporal and spatial changes in cohesin localization during early prophase in wild-type Drosophila ovaries as well as in mutants with compromised cohesion and/or homologous recombination. Drosophila oogenesis provides a unique opportunity to examine important changes in chromosome morphology that occur during meiotic prophase. Because not all germ-line cells adopt an oocyte fate, the 16-cell cyst allows direct comparison of nuclei that assemble meiotic chromosome cores (and SC) with those that do not. Importantly, this dynamic transformation in chromosome structure depends upon and must occur within the context of functional sister-chromatid cohesion. In addition, these events are crucial for homologous recombination and, therefore, accurate segregation of meiotic chromosomes (Khetani, 2007).

Analysis of chromosome spread preparations from wild-type ovaries indicates that the cohesin subunits SMC1 and SMC3 localize along the arms of chromosomes in all 16 cells of each cyst. However, thread-like cohesin SMC staining is only observed in the nuclei that build a SC. The simplest model to explain the differences observed in pro-nurse cells and pro-oocytes is that multiple cohesin complexes come together to form long continuous threads of cohesin staining in nuclei that build SC. It is proposed that chromosomes in pro-nurse cells maintain an extended interphase-like organization in which cohesin complexes localize along the arms but fail to assemble into this higher order structure. By contrast, the formation of chromosome cores in a subset of nuclei occurs when multiple cohesin complexes along the arms coalesce into threads and, thereby, bring about the shortening of the longitudinal axes of meiotic chromosomes (Khetani, 2007).

Formation of chromosome cores represents the first step in the organized assembly of the SC. Although DSBs are required for synapsis of homologues in a number of species, lateral elements are still visible in mutants that fail to make DSBs and therefore lack tripartite SC. Moreover, chromosome cores have been proposed to serve as the scaffold upon which SC components organize and assemble. Genetic and cytological analyses in a number of organisms have confirmed that the cohesin complex plays an integral role in SC assembly. This work argues that cohesin SMCs as well as the cohesion protein ORD are stable components of meiotic chromosome cores, which remain intact when DNase I treatment removes chromatin loops. Interestingly, in chromosome spread preparations, localization of cohesin subunits and ORD appears to be restricted to chromosome cores; diffuse staining is not detectable in the areas between threads. These data are consistent with the model that, in Drosophila meiotic cells, cohesion proteins localize predominantly along the chromatid axes and are not found decorating the loops. Similar arguments have been made for cohesin localization in S. cerevisiae and mouse meiotic cells (Khetani, 2007).

Evidence is provided that the meiosis-specific protein, C(2)M, is required for chromosome core formation in Drosophila oocytes. Chromosome spread preparations indicate that in the absence of C(2)M activity, SMC1 and SMC3 diffuse staining is visible throughout the nuclei of all 16 cells within each cyst, indicative of the association of cohesin subunits with chromosome arms. However, no thread-like SMC1/3 or ORD staining is observed in c(2)M-mutant germaria. These data indicate that C(2)M protein controls an early step in the formation of chromosome cores. It is proposed that, by virtue of its ability to interact with cohesin SMC proteins (Heidmann, 2004), C(2)M drives the association of cohesin complexes to form meiotic chromosome cores. Failure in this process would prohibit subsequent assembly of the SC in c(2)M mutant germaria as evidenced by lack of thread-like immunostaining for the transverse filament protein C(3)G (Manheim, 2003). These results also are consistent with EM localization of C(2)M protein along the lateral elements of the wild-type Drosophila SC (Anderson, 2005; Khetani, 2007).

Surprisingly, it was found that the cohesion protein ORD is required for the maintenance of chromosome cores during early pachytene in Drosophila. In the absence of ORD activity, thread-like SMC1/3 staining is visible in region 2A of the germarium; however, the intensity and integrity of SMC1/3 threads deteriorate as pachytene progresses within the germarium. Analysis of chromosome spread preparations indicates that, although chromosome cores disassemble, the cohesin subunits SMC1 and SMC3 remain associated with the chromosome arms in ord^null germaria (Khetani, 2007).

Quantitative analyses of temporal progression of SMC1/3 and C(3)G defects in ord mutant germaria (Webber, 2004), as well as co-immunostaining experiments that simultaneously monitored cohesin subunits and C(3)G in individual ord^null nuclei, argue that the onset of chromosome core dissolution precedes fragmentation of thread-like epifluorescent signal for the SC marker, C(3)G. These data demonstrate that initial assembly of cores is not sufficient for stable SC; instead, maintenance of chromosome cores is an ongoing requirement to preserve SC integrity (Khetani, 2007).

Why do cores disassemble in the absence of ORD activity? By immunofluorescence, continuous thread-like C(2)M and C(3)G staining is transiently present during early pachytene in ord^null germaria; however, at the same stage, normal tripartite SC is not detectable by EM (Webber, 2004). Therefore, although the highly organized SC ultrastructure is absent, some aspects of SC assembly still occur in the absence of ORD function [namely recruitment of C(2)M and C(3)G]. These data suggest that ORD is required to recruit additional proteins along the chromosome cores and/or lateral/axial elements that are required for core integrity. Alternatively, ORD itself might be required to maintain C(2)M-mediated organization of the cores into stable structures (Khetani, 2007).

Programmed cycles of stress and relaxation along meiotic chromosomes have been proposed to govern several critical events during prophase. One possibility is that in the absence of ORD function – although chromosome cores assemble – they are unable to withstand normal changes in compression and/or relaxation, and subsequently buckle. Interestingly, it has been reported that, during wild-type pachytene, the SC shortens significantly as cysts move through the Drosophila germarium. If chromosome cores that assemble in the absence of ORD are inherently unstable, programmed shortening of the SC could cause additional stress that results in fragmentation of the cores. Curiously, it has been observed that in later stages (stages 3-6 of the vitellarium), continuous thread-like SMC1/3 staining often reappears in ord^null oocytes. These stages loosely correspond to the time after which the SC reaches its shortest length and starts to expand in wild type. Chromosome cores might be able to reassemble at these later stages in ord^null ovarioles if decompaction of the chromosome axes reduces stress (Khetani, 2007).

Breakdown of chromosome cores in ord^null germaria is also temporally linked to the onset of DSBs. Both the timing (early 2A) and the number of DSBs are normal in ord^null germaria (Webber, 2004). However, fragmentation of SMC1/3 thread-like staining is detected in late region 2A, after the onset of DSBs. Therefore, induction of DSBs might contribute to destabilization of chromosome cores that are compromised due to lack of ORD protein. A similar model has been proposed to explain the phenotypes associated with disruption of the Pds5 orthologue (Spo76) in Sordaria (Storlazzi, 2003). Although AEs are continuous during early prophase I in spo76-1 mutants, they fragment prematurely in a DSB-dependent fashion (Khetani, 2007).

The disassembly of chromosome cores in ord^null germaria is most probably responsible for the severe reduction in crossovers between homologues in mutant females. ORD activity is essential for the crucial decision each chromatid must make after the induction of DSBs – namely whether the broken chromatid will choose its sister or its homologue for repair. Strand invasion and crossovers are biased towards the homologue during meiosis, resulting in stable chiasmata that keep homologous chromosomes physically associated until anaphase I. Previous experiments have shown that ORD activity is required for homologue bias during meiotic recombination in Drosophila. In ord^null females, the frequency of crossovers between homologues is decreased, while that between sister chromatids is significantly increased (Webber, 2004). These data combined with current analyses suggest that chromosome cores are necessary for homologue bias during meiosis, and partner choice takes place in late region 2A or region 2B of the Drosophila germarium (Khetani, 2007).

In ord^null germaria, SMC1 and SMC3 fail to accumulate at centromeres, but appear to localize normally along chromosome arms within all 16 cells of each germ-line cyst. These results suggest that distinct pathways mediate cohesin loading on the arms and centromeres during Drosophila meiosis. It cannot be differentiated whether centromeric loading of SMC1 and SMC3 is completely ablated or whether cohesin SMCs are able to load at centromeres but are quickly removed when ORD activity is absent. Regardless, accumulation of cohesin subunits at the centromeres of meiotic chromosomes appears to depend on ORD function. By contrast, ORD activity is not required for stable association of SMC1 and SMC3 along chromosome arms. Curiously, after germ-line cells adopt a nurse cell fate, ORD is no longer necessary for centromeric accumulation of cohesin; the clustered finger-like projections of SMC1/3 staining at nurse cell centromeres in ord^null ovarioles (stages 3-4) are indistinguishable from that in wild type. However, even at these later stages when cohesin subunits are visible at nurse cell centromeres, SMC1 or SMC3 are never detected at the centromeres of oocytes in ord^null ovarioles. Therefore, the data implicate ORD in a meiosis-specific pathway for cohesin loading and/or accumulation at centromeres (Khetani, 2007).

Co-immunostaining experiments with CID and SMC1/3 antibodies indicate that absence of cohesin subunits appears to be restricted to the centromeres in ord^null germaria; within the resolution limits of the chromosome spread images, the area lacking SMC1/3 signal is approximately the same size as the CID staining. These data suggest that cohesin loading and/or accumulation at pericentromeric heterochromatin occurs in the absence of ORD function. However, the striking enrichment of SMC1/3 in pericentromeric heterochromatin prominent in wild type is not observed. Therefore, the analysis of defects of cohesin localization in ord^null germaria suggest that normal loading and/or accumulation of cohesin is regulated differently even within the different domains of heterochromatin in and around centromeres. In addition, the chromocenter appears to be less stable in ord^null oocytes, raising the possibility that changes in heterochromatin structure in the absence of ORD activity diminishes the ability of centromeres to associate (Khetani, 2007).

Chromosome spread experiments clearly indicate that SMC1 and SMC3 are stably associated with the chromosome arms of both pro-nurse cells and pro-oocytes within ord^null germaria. However, whether the localization of cohesin subunits represents functional cohesin is not clear. From genetic and cytological studies, it is known that meiotic cohesion is completely absent in ord^null oocytes by the time that meiotic chromosomes make microtubule attachments (Bickel, 2002; Bickel, 1997). Separated sister chromatids have not been detected during early pachytene in ord^null germaria by FISH (Webber, 2004). However, catenation might hold sisters together at this time, thereby masking defects arising from the absence of cohesin-mediated cohesion (Khetani, 2007).

Stepwise loading of cohesin subunits during meiotic prophase has been described for a number of organisms. In worms and grasshoppers, stable association of cohesin SMCs in the absence of non-SMC subunits has been reported for meiotic chromosomes. One possibility is that, in the absence of ORD, cohesin SMCs load without their non-SMC partners. Another possibility is that the entire cohesin complex loads in ord^null ovaries but cohesin-mediated cohesion is not established. At least two reports have indicated that, in S. cerevisiae, the binding of cohesin to specific genomic locations is insufficient for cohesin-mediated cohesion. In addition, recent work has been elegantly demonstrated that different populations of chromatin-bound mitotic cohesin exist within Hela cells. This work suggests that, during replication and the establishment of cohesion, a subset of chromatin-bound cohesin complexes is converted from 'dynamically associated' to 'irreversibly bound'. ORD might be necessary for the establishment of meiotic sister-chromatid cohesion but not for association of cohesin subunits with the chromosomes. Alternatively, cohesion might be established in the absence of ORD activity but not maintained (Khetani, 2007).

In most species, a meiosis-specific α-kleisin subunit (Rec8) promotes meiotic cohesion by interacting with the heads of the SMC subunits and closing the cohesin ring. Surprisingly, an obvious Rec8 orthologue has not been identified in the Drosophila genome. Although its limited sequence homology to Rec8 in other organisms has led to the proposal that C(2)M functions as the meiosis-specific α-kleisin subunit of the cohesin complex in Drosophila (Schleiffer, 2003), phenotypic analysis of c(2)M mutant flies is inconsistent with this hypothesis (Manheim, 2003). In contrast to other Drosophila mutations that disrupt meiotic cohesion, defects of meiosis II segregation are negligible in c(2)M females and accurate chromosome segregation during male meiosis also does not depend on C(2)M function (Manheim, 2003). Moreover, female germ-line expression of a mutated C(2)M transgene in which putative separase cleavage sites were disrupted did not result in meiotic segregation defects. Finally, localization of C(2)M protein in whole-mount preparations (Manheim, 2003) indicates that, like C(3)G protein, C(2)M is restricted to the subset of cells within each germ-line cyst that build SC. Therefore, it is proposed that the kleisin domain in C(2)M allows it to interact with cohesin SMC subunits and that C(2)M plays an essential role in building meiotic chromosome cores, but does not promote meiotic cohesion (Khetani, 2007).

In several respects, the behavior of ORD protein is consistent with it performing the role of Rec8 during Drosophila meiosis. Null mutations in ord eliminate meiotic cohesion in both sexes and ORD colocalizes extensively with cohesin SMC subunits during early pachytene in females. During male meiosis, ORD remains associated with spermatoctye centromeres throughout prophase I (E. M. Balicky, Regulation of chromosome segregation by ORD and dRING during Drosophila meiosis, PhD thesis, Dartmouth College, Hanover, NH, 2004) and is not lost until anaphase II when centromeric cohesion is released (Balicky, 2002). Moreover, retention of ORD at centromeres until anaphase II depends on the activity of the Drosophila Shugoshin ortholog, Mei-S332 (E. M. Balicky, PhD thesis, Dartmouth College, Hanover, NH, 2004). It has not been possible to detect ORD (or cohesin SMCs) on meiotic chromosomes during late oogenesis; however, given that ORD is required for cohesion in both sexes and localizes to spermatocyte centromeres until anaphase II, it seems likely that lack of signal in mature oocytes is due to antibody accessibility issues and/or detection limitations, not the absence of the protein (Khetani, 2007).

Although several pieces of data are consistent with the model that ORD protein provides Rec8 activity in Drosophila, the size of ORD protein (479 amino acids) may be too small to bridge the heads of the Drosophila SMC1/3 dimer. In addition, ORD does not share obvious sequence homology with Rec8, Scc3/SA or regulators of cohesin (Pds5, Scc4, Scc2). One possibility is that during Drosophila meiosis, two proteins collaborate to provide Rec8 function. Such is the case for Drosophila separase, which is composed of two subunits encoded by separate genes. ORD may cooperate with Rad21 or another unidentified protein to provide Rec8 function during meiosis. Why flies would use an altered mechanism to accomplish such a highly conserved activity is an enigma. However, further analysis of the regulation of meiotic cohesion in Drosophila should provide important evolutionary insights into fundamental aspects of recombination and chromosome segregation during meiosis (Khetani, 2007).

The cohesion protein ORD is required for homologue bias during meiotic recombination

During meiosis, sister chromatid cohesion is required for normal levels of homologous recombination, although how cohesion regulates exchange is not understood. Null mutations in orientation disruptor (ord) ablate arm and centromeric cohesion during Drosophila meiosis and severely reduce homologous crossovers in mutant oocytes. ORD protein localizes along oocyte chromosomes during the stages in which recombination occurs. Although synaptonemal complex (SC) components initially associate with synapsed homologues in ord mutants, their localization is severely disrupted during pachytene progression, and normal tripartite SC is not visible by electron microscopy. In ord germaria, meiotic double strand breaks appear and disappear with frequency and timing indistinguishable from wild type. However, Ring chromosome recovery is dramatically reduced in ord oocytes compared with wild type, which is consistent with the model that defects in meiotic cohesion remove the constraints that normally limit recombination between sisters. It is concluded that ORD activity suppresses sister chromatid exchange and stimulates inter-homologue crossovers, thereby promoting homologue bias during meiotic recombination in Drosophila (Webber, 2004; full text of article).

Although sister chromatid cohesion during meiosis is essential for normal levels of homologous recombination, the mechanism by which cohesion regulates meiotic recombination has remained elusive. This study provides evidence that meiotic cohesion is required to suppress sister chromatid exchange and thereby promote inter-homologue recombination during meiosis. Moreover, the link is further defined between meiotic cohesion and the formation/stabilization of the SC by focusing analysis on a cohesion protein that is distinct from the cohesin complex (Webber, 2004).

The localization of ORD protein along the entire length of oocyte chromosomes is consistent with previous genetic and cytological evidence that ORD is essential for arm and centromeric cohesion during female meiosis. Bright centromeric ORD foci in the premeiotic cysts of region 1 are similar to those detected in the mitotic cysts within the testis, and diffuse ORD signal throughout the nuclei of some 8-cell cysts and all 16-cell cysts suggests that ORD accumulates along the length of chromosomes before premeiotic S phase. Unlike SC components, ORD protein is not restricted to a subset of nuclei within the cyst. In chromosome spreads, ORD linear elements are visible in all of the cells within the cyst. However, distinct stretches of ribbonlike ORD staining are more pronounced within nuclei that are assembling SC, perhaps because of the high degree of chromatin structure/organization within the context of the SC. In addition, SC compaction and shortening during pachytene probably enhance the ribbonlike nature of the ORD signal in the pro-oocytes. Like the SC components C(3)G and C(2)M, ORD remains along the entire length of oocyte bivalents throughout pachytene. These data are consistent with the model that ORD promotes arm and centromeric cohesion during the stages in which meiotic recombination occurs (Webber, 2004).

This work indicates that ORD activity is required for normal SC. Although C(3)G and C(2)M appear to localize normally during early prophase in ord mutants, their association with bivalents is severely disrupted during pachytene progression. By EM analysis, SC-like structures are observed in ord^null germaria that appear to be composed of CEs without distinct axial/lateral elements (AEs/LEs) of the SC assemble. These results, coupled with fluorescent immunodetection of C(3)G in ord mutants, suggest that some CE components can assemble in the absence of normal AE/LEs. Although somewhat unexpected, similar results have been obtained in yeast and mice and have led to the proposal that CEs form in the absence of AE/LEs by using the cohesin complex core as a scaffold for assembly. Interestingly, although cohesin subunits initially associate with chromatid arms in ord germaria, their localization starts to deteriorate before the time that C(3)G localization defects are first seen. Therefore, assembly of CEs in ord mutants may rely on the cohesin complex and disintegrate when cohesin localization is destabilized (Webber, 2004).

The results are consistent with the hypothesis that ORD activity is required for the formation of AE/LEs during meiosis in Drosophila. One possibility is that ORD is a structural component of the AE/LEs. However, this is thought unlikely because ORD is essential for meiotic sister chromatid cohesion in both males and females, and Drosophila spermatocytes do not undergo meiotic recombination or form SC. In addition, the observation that ribbonlike ORD signal is visible in all 16 cells of germarial cysts demonstrates that chromatin association of ORD occurs in cells that never form extensive SC. Therefore, it is proposed that ORD activity is a prerequisite for formation of the AE/LE and may colocalize with this structure, but that the primary function of ORD protein is to promote sister chromatid cohesion (Webber, 2004).

During meiosis, only crossovers between homologous chromosomes can generate a stable chiasma that will promote proper segregation during meiosis I. After the formation of a DSB, strand invasion into the sister chromatid will not be productive in maintaining the association of homologous chromosomes. Physical evidence suggests that recombination is biased in S. cerevisiae meiosis to favor recombination between homologues. Although crossovers between sisters are not completely inhibited, homologues are the preferred partners and inter-homologue intermediates represent ~70% of the joint molecules that form during strand invasion. When the AE/LE component Red1p is missing/mutated, the percentage of inter-sister events increases, indicating that in yeast, normal AE/LEs suppress sister chromatid exchange (Webber, 2004).

Although recombination intermediates cannot be analyzed at the molecular level in Drosophila, a Ring chromosome genetic assay argues that mutations in ord disrupt homologue bias during Drosophila meiosis. Similar to other investigators, a Ring/Rod ratio in wild type was obtained that is <1 and probably reflects the level of sister chromatid exchange that normally occurs in Drosophila oocytes. However, the recovery of Ring chromosome–containing progeny from ord⁶/ord¹⁰ and ord⁵/ord¹⁰ females is significantly lower than from ord⁺ females, and ord^null females exhibit lower transmission of the Ring chromosome than those with partial ORD activity (Webber, 2004).

The results are most consistent with the model that disruption of ORD activity allows increased levels of sister chromatid exchange during meiosis. Null mutations in ord cause random segregation of normal Rod chromosomes due to complete loss of centromeric cohesion. Therefore, reduced recovery of the Ring chromosome in ord⁵/ord¹⁰ females cannot arise because the Ring chromosome is more sensitive to cohesion defects than the Rod chromosome. Moreover, nearly equivalent recovery of Ring and Rod chromosomes from c(3)G oocytes argues that cis-acting sequences on the R(1)2 chromosome are capable of mediating proper centromeric cohesion and kinetochore function during meiosis. Although failure to decatenate interlocked Ring chromosomes could result in their reduced recovery, chromosome bridges have not been observed for normal chromosomes in ord mutants. Therefore, it is proposed that ORD activity is required during Drosophila meiosis to limit exchange between sisters and thereby promote inter-homologue crossovers. These data represent the first evidence in metazoans of a gene product that suppresses recombination between sister chromatids during meiosis (Webber, 2004).

An increase in sister exchange in ord mutants cannot be explained by the disruption of C(3)G localization that occurs during pachytene progression. Although inter-homologue crossovers are abolished in c(3)G mutant females. This study found DSBs are not significantly reduced in c(3)G oocytes. Therefore, absence of the SC CE is not sufficient to lift the constraints that limit inter-sister events. Similar argument have been made for Caenorhabditis elegans meiosis (Webber, 2004).

Homologue bias during meiotic recombination in Drosophila most likely arises from mechanisms that promote homologous recombination as well as those that suppress inter-sister events. Recombination defects in ord mutants may result from disruption of both pathways. It is proposed that the absence of ORD activity disrupts the formation and/or stability of AE/LEs which normally limit inter-sister strand invasion after the induction of DSBs. Although meiotic DSBs appear and disappear with normal frequency and timing in ord^null germaria, the preferred pathway for DSB repair in ord oocytes is strand invasion into the sister chromatid. In addition, ORD is required to maintain C(3)G localization and presumably the integrity of the CE. Elimination of crossovers in c(3)G mutants indicates that the SC is absolutely required for repair of DSBs as inter-homologue crossovers in Drosophila. Although crossovers are decreased in oocytes completely lacking ORD activity, they are not abolished. Transient localization of C(3)G in ord mutants may allow the few crossovers that do occur, but subsequent disruption of C(3)G localization most likely prevents normal numbers of inter-homologue events. It is concluded that ORD activity promotes homologue bias during Drosophila meiosis by suppressing inter-sister events as well as promoting inter-homologue events and that disruption of both pathways leads to decreased numbers of crossovers in ord mutants (Webber, 2004).

Meiosis-specific controls that direct partner choice during recombination are essential for accurate chromosome segregation of homologous chromosomes during meiosis I. The results provide critical answers about how sister chromatid cohesion ensures that crossovers occur between homologous chromosomes during meiosis in Drosophila. Because cohesion is required for normal levels of meiotic exchange in several species, it is predicted that this mechanism is highly conserved among eukaryotes (Webber, 2004).

A proposed role for the Polycomb group protein dRING in meiotic sister-chromatid cohesion

ORD protein is required for accurate chromosome segregation during male and female meiosis in Drosophila melanogaster. Null ord mutations result in random segregation of sister chromatids during both meiotic divisions because cohesion is completely abolished prior to kinetochore capture of microtubules during meiosis I. Previous analyses of mutant ord alleles have led to a proposal that the C-terminal half of the ORD protein mediates protein-protein interactions that are essential for sister-chromatid cohesion. To identify proteins that interact with ORD, a yeast two-hybrid screen was conducted using an ORD bait and isolated dRING, a core subunit of the Drosophila Polycomb repressive complex 1. A missense mutation in ORD completely ablates the two-hybrid interaction with dRING and prevents nuclear retention of the mutant ORD protein in male meiotic cells. Using affinity-purified antibodies generated against full-length recombinant dRING, it is demonstrated that dRING protein is expressed in the male and female gonads and colocalizes extensively with ORD on the chromatin of primary spermatocytes during G2 of meiosis. These results suggest a novel role for the Polycomb group protein dRING and are consistent with the model that interaction of dRING and ORD is required to promote the proper segregation of meiotic chromosomes (Balicky, 2004).

Genetic interactions between mei-S332 and ord in the control of sister-chromatid cohesion

The Drosophila mei-S332 and ord gene products are essential for proper sister-chromatid cohesion during meiosis in both males and females. Flies were constructed that contain null mutations for both genes. Double-mutant flies are viable and fertile. Therefore, the lack of an essential role for either gene in mitotic cohesion cannot be explained by compensatory activity of the two proteins during mitotic divisions. Analysis of sex chromosome segregation in the double mutant indicates that ord is epistatic to mei-S332. ord is not required for Mei-S332 protein to localize to meiotic centromeres. Although overexpression of either protein in a wild-type background does not interfere with normal meiotic chromosome segregation, extra ORD⁺ protein in mei-S332 mutant males enhances nondisjunction at meiosis II. These results suggest that a balance between the activity of mei-S332 and ord is required for proper regulation of meiotic cohesion and demonstrate that additional proteins must be functioning to ensure mitotic sister-chromatid cohesion (Bickel, 1998).

Mechanisms of chromosome orientation revealed by two meiotic mutants in Drosophila

Two disjunction defective meiotic mutants, ord and mei-S332, each of which disrupts meiosis in both male and female Drosophila melanogaster, were analyzed cytologically and genetically in the male germ-line. It was observed that sister-chromatids are frequently associated abnormally during prophase I and metaphase I in ord. Sister chromatid associations in mei-S332 are generally normal during prophase I and metaphare I. By telophase I, sister chromatids have frequently precociously separated in both mutants. During the first division sister chromatids disjoin from one another frequently in ord and rarely in mei-S332. It is argued that the simplest interpretation of the observations is that each mutant is defective in sister chromatid cohesiveness and that the defect in ord manifests itself earlier than does the defect in mei-S332. In addition, based on these mutant effects, several conclusions regarding normal meiotic processes are drawn. (1) The phenotype of these mutants support the proposition that the second meiotic metaphase (mitotic-type) position of chromosomes and their equational orientation is a consequence of the equilibrium, at the metaphase plate, of pulling forces acting at the kinetochores and directed towards the poles. (2) Chromosomes which lag during the second meiotic division tend to be lost. (3) Sister chromatid cohesiveness, or some function necessary for sister chromatid cohesivenss, is required for the normal reductional orientation of sister kinetochores during the first meiotic division. (4) The kinetochores of a half-bivalent are double at the time of chromosome orientation during the first meiotic division. Finally, functions which are required throughout meiosis in both sexes must be considered in the pathways of meiotic control (Goldstein, 1980).

Search PubMed for articles about Drosophila Ord

Anderson, L. K., Royer, S. M., Page, S. L., McKim, K. S., Lai, A., Lilly, M. A. and Hawley, R. S. (2005). Juxtaposition of C(2)M and the transverse filament protein C(3)G within the central region of Drosophila synaptonemal complex. Proc. Natl. Acad. Sci. 102: 4482-4487. PubMed citation: 15767569

Balicky, E. M., Endres, M. W., Lai, C. and Bickel, S. E. (2002). Meiotic cohesion requires accumulation of ORD on chromosomes before condensation. Mol. Biol. Cell 13(11): 3890-900. PubMed citation: 12429833

Balicky, E. M., Young, L., Orr-Weaver, T. L. and Bickel, S. E. (2004). A proposed role for the Polycomb group protein dRING in meiotic sister-chromatid cohesion. Chromosoma 112(5): 231-9. 14669021

Bickel, S. E., Wyman, D. W., Miyazaki, W. Y., Moore, D. P. and Orr-Weaver, T. L. (1996). Identification of ORD, a Drosophila protein essential for sister chromatid cohesion. EMBO J. 15(6): 1451-9. PubMed citation: 8635478

Bickel, S. E., Wyman, D. W. and Orr-Weaver, T. L. (1997). Mutational analysis of the Drosophila sister-chromatid cohesion protein ORD and its role in the maintenance of centromeric cohesion. Genetics 146(4): 1319-31. PubMed citation: 9258677

Bickel, S. E., Moore, D. P., Lai, C. and Orr-Weaver, T. L. (1998). Genetic interactions between mei-S332 and ord in the control of sister-chromatid cohesion. Genetics 150(4): 1467-76. 9832524

Bickel, S. E., Orr-Weaver, T. L. and Balicky, E. M. (2002). The sister-chromatid cohesion protein ORD is required for chiasma maintenance in Drosophila oocytes. Curr. Biol. 12(11): 925-9. PubMed citation: 12062057

Goldstein, L. S. (1980). Mechanisms of chromosome orientation revealed by two meiotic mutants in Drosophila melanogaster. Chromosoma 78: 79111. Medline abstract: 6769652

Heidmann, D., Horn, S., Heidmann, S., Schleiffer, A., Nasmyth, K. and Lehner, C. F. (2004). The Drosophila meiotic kleisin C(2)M functions before the meiotic divisions. Chromosoma 113: 177-187. PubMed citation: 15375666

Khetani, R. S. and Bickel, S. E. (2007). Regulation of meiotic cohesion and chromosome core morphogenesis during pachytene in Drosophila oocytes. J. Cell Sci. 120(Pt 17): 3123-37. PubMed citation: 17698920

Lee, J. Y. and Orr-Weaver, T. L. (2001). The molecular basis of sister-chromatid cohesion. Annu. Rev. Cell Dev. Biol. 17: 753-777. PubMed citation: 11687503

Manheim, E. A. and McKim, K. S. (2003). The synaptonemal complex component C(2)M regulates meiotic crossing over in Drosophila. Curr. Biol. 13: 276-285. PubMed citation: 12593793

Mason, J. M. (1976). Orientation disruptor (ord): a recombination-defective and disjunction-defective meiotic mutant in Drosophila melanogaster. Genetics 84: 545-572. PubMed citation: 826453

Miyazaki, W. Y. and Orr-Weaver, T. L. (1992). Sister-chromatid misbehavior in Drosophila ord mutants. Genetics 132: 1047-1061. PubMed citation: 1459426

Nasmyth, K. (2002). Segregating sister genomes: the molecular biology of chromosome separation. Science 297: 559-565. PubMed citation: 12142526

Page, S. L. and Hawley, R. S. (2001). c(3)G encodes a Drosophila synaptonemal complex protein. Genes Dev. 15: 3130-3143. PubMed citation: 11731477

Petronczki, M., Siomos, M. F. and Nasmyth, K. (2003). Un Menage a Quatre: the molecular biology of chromosome segregation in meiosis. Cell 112: 423-440. PubMed citation: 12600308

Revenkova, E., Eijpe, M., Heyting, C., Hodges, C. A., Hunt, P. A., Liebe, B., Scherthan, H. and Jessberger, R. (2004). Cohesin SMC1 beta is required for meiotic chromosome dynamics, sister chromatid cohesion and DNA recombination. Nat. Cell Biol. 6: 555-562. PubMed citation: 15146193

Schleiffer, A., Kaitna, S., Maurer-Stroh, S., Glotzer, M., Nasmyth, K. and Eisenhaber, F. (2003). Kleisins: a superfamily of bacterial and eukaryotic SMC protein partners. Mol. Cell 11: 571-575. PubMed citation: 12667442

Shintomi, K. and Hirano, T. (2007). How are cohesin rings opened and closed? Trends Biochem Sci. 32: 154-157. PubMed citation: 1732040

Stack, S. M. and Anderson, L. K. (2001). A model for chromosome structure during the mitotic and meiotic cell cycles. Chromosome Res. 9: 175-198. PubMed citation: 11330393

Storlazzi, A., Tesse, S., Gargano, S., James, F., Kleckner, N. and Zickler, D. (2003). Meiotic double-strand breaks at the interface of chromosome movement, chromosome remodeling, and reductional division. Genes Dev. 17: 2675-2687. PubMed citation: 14563680

Webber, H. A., Howard, L. and Bickel, S. E. (2004). The cohesion protein ORD is required for homologue bias during meiotic recombination. J. Cell Biol. 164(6): 819-29. PubMed citation: 15007062

date revised: 27 April 2008

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