InteractiveFly: GeneBrief

orientation disrupter: Biological Overview | References

Gene name - orientation disrupter

Synonyms -

Cytological map position - 59D4-59D4

Function - cohesion protein

Keywords - oocyte, spermatocyte, meiotic chromosome and centromeric cohesion, pachytene progression, homolog biased crossovers, suppression of sister chromatid exchange, rapidly evolving gene

Symbol - ord

FlyBase ID: FBgn0003009

Genetic map position - 2R:19,158,778..19,161,798 [-]

Classification - novel protein conserved only in Drosophila species but not in Anopheles or Apis

Cellular location - nuclear

NCBI link: EntrezGene

Ord orthologs: Biolitmine
Recent literature
Gyuricza, M. R., Manheimer, K. B., Apte, V., Krishnan, B., Joyce, E. F., McKee, B. D. and McKim, K. S. (2016). Dynamic and stable cohesins regulate synaptonemal complex assembly and chromosome segregation. Curr Biol [Epub ahead of print]. PubMed ID: 27291057
Assembly of the synaptonemal complex (SC) in Drosophila depends on two independent pathways defined by the chromosome axis proteins C(2)M and ORD. Because C(2)M encodes a Kleisin-like protein and ORD is required for sister-chromatid cohesion, the hypothesis was tested that these two SC assembly pathways depend on two cohesin complexes. Through single- and double-mutant analysis to study the mitotic cohesion proteins Stromalin (SA) and Nipped-B (SCC2) in meiosis, evidence was provided that there are at least two meiosis-specific cohesin complexes. One complex depends on C(2)M, SA, and Nipped-B. Despite the presence of mitotic cohesins SA and Nipped-B, this pathway has only a minor role in meiotic sister-centromere cohesion and is primarily required for homolog interactions. C(2)M is continuously incorporated into pachytene chromosomes even though SC assembly is complete. In contrast, the second complex, which depends on meiosis-specific proteins SOLO, SUNN, and ORD is required for sister-chromatid cohesion, localizes to the centromeres and is not incorporated during prophase. Multiple cohesin complexes may provide the diversity of activities required by the meiotic cell.

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 ordnull 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, smc1exc46, 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 (ordnull) 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 ordnull 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. ordnull 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 ordnull 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 ordnull 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 ordnull 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 ordnull 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 ordnull 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 ordnull germaria (Khetani, 2007).

To characterize the progressive deterioration of SMC1 and SMC3 thread-like staining in ordnull 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 ordnull 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 ordnull oocyte nuclei contained no visible SMC1 staining and 53% contained severely fragmented threads. SMC3 signal was undetectable in approximately 52% of region 3 ordnull 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 ordnull 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 ordnull 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 ordnull 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 ordnull 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 ordnull oocytes: The progressive deterioration of chromosome cores in ordnull 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 ordnull 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 ordnull germaria (Khetani, 2007).

Continuous thread-like staining is evident for both C(3)G and SMC1/3 in early region 2A nuclei of ordnull 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)MEP[2115] (Manheim, 2003). To observe ORD localization, a c(2)MEP[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)MEP[2115];P{GFP-ORD} females (24.44%, n=753) was similar to that previously reported for c(2)MEP[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)MEP[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 ordnull 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 ordnull 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 ordnull 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 ordnull 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 ordnull ovarioles if decompaction of the chromosome axes reduces stress (Khetani, 2007).

Breakdown of chromosome cores in ordnull germaria is also temporally linked to the onset of DSBs. Both the timing (early 2A) and the number of DSBs are normal in ordnull 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 ordnull 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 ordnull 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 ordnull 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 ordnull 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 ordnull 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 ordnull 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 ordnull 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 ordnull 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 ordnull 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 ordnull 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 ordnull 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 ordnull 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).

A pathway for synapsis initiation during zygotene in Drosophila oocytes

Formation of the synaptonemal complex (SC), or synapsis, between homologs in meiosis is essential for crossing over and chromosome segregation. How SC assembly initiates is poorly understood but may have a critical role in ensuring synapsis between homologs and regulating double-strand break (DSB) and crossover formation. This study investigated the genetic requirements for synapsis in Drosophila and found that there are three temporally and genetically distinct stages of synapsis initiation. In meiotic prophase 1 'early zygotene' oocytes, synapsis is only observed at the centromeres. It was also found that nonhomologous centromeres are clustered during this process. In 'mid-zygotene' oocytes, SC initiates at several euchromatic sites. The centromeric and first euchromatic SC initiation sites depend on the cohesion protein ORD. In 'late zygotene' oocytes, SC initiates at many more sites that depend on the Kleisin-like protein C(2)M. Surprisingly, late zygotene synapsis initiation events are independent of the earlier mid-zygotene events, whereas both mid and late synapsis initiation events depend on the cohesin subunits SMC1 and SMC3. It is proposed that the enrichment of cohesion proteins at specific sites promotes homolog interactions and the initiation of euchromatic SC assembly independent of DSBs. Furthermore, the early euchromatic SC initiation events at mid-zygotene may be required for DSBs to be repaired as crossovers (Tanneti, 2011).

Drosophila pro-oocytes develop within 16-cell cysts that are arranged in temporal order within the ovary. Each ovary contains several germaria, where pairs of pro-oocytes begin their development and enter prophase in region 2a and a single oocyte is selected by region 3. Oocytes are defined by the presence of the synaptonemal complex (SC), which is detected by antibodies to the transverse element C(3)G (Page, 2001), a coiled-coil protein similar to proteins in budding yeast (ZIP1), C. elegans (SYP-1, SYP-2), and mammals (SYCP1) (Page, 2004; Watts, 2011). Zygotene pro-oocytes were identified by their patchy C(3)G staining, as opposed to the thread-like staining typical of pachytene. Furthermore, by comparing the amount of synapsis to the relative positions of the pro-oocytes in the wild-type germarium, three stages of zygotene were defined (Tanneti, 2011).

First, early zygotene pro-oocytes have one or two patches of C(3)G that colocalize with CID, a centromere-specific histone H3. These pro-oocytes reside in the earliest (most anterior) part of region 2a, indicating that synapsis initiates at the centromeres before any other sites. These results were confirmed by comparing CID localization to histone modifications specific for the heterochromatin or euchromatin. Because there are four pairs of centromeres, the observation that most wild-type pro-oocytes have one or two CID foci indicates that nonhomologous centromeres cluster in meiotic prophase, confirming previous observations using electron microscopy (Tanneti, 2011).

Second, mid-zygotene pro-oocytes have the centromeric C(3)G staining plus approximately six additional sites in the euchromatin. Finally, late zygotene pro-oocytes contain many C(3)G foci but lack the continuous threadlike pattern of pachytene. Surprisingly, the mid-zygotene patches do not appear to get longer. Instead, there are more patches in late zygotene, suggesting that the progression from mid- to late zygotene involves the establishment of new SC initiation sites rather than polymerization from the small number of sites in mid-zygotene. It is suggested that the noncentromeric C(3)G sites in mid-zygotene represent the first euchromatic sites to initiate synapsis. This study provides evidence that the mid-zygotene sites have features in common with centromere synapsis sites but are mechanistically distinct and genetically separable from the additional synapsis initiation sites observed in late zygotene (Tanneti, 2011).

C(2)M is a lateral element component and is a member of the Kleisen family that includes Rec8 and Rad21 homologs (Schleiffer, 2003). In wild-type, C(2)M colocalizes with C(3)G in most locations except at the centromeres. In females lacking C(2)M, the first two stages of zygotene appear to occur normally. Early zygotene pro-oocytes exhibit one or two foci of CID that colocalize with C(3)G, showing that C(2)M is not required for centromere clustering or centromere synapsis. These results confirm previous observations (Khetani, 2007) that C(2)M is not required for centromere clustering in pachytene oocytes and are consistent with the observation that C(2)M does not localize to the centromeric regions. Early zygotene in c(2)M mutants is followed by cysts with several patches of euchromatic C(3)G staining that resemble wildtype cells in mid-zygotene. Synapsis in a c(2)M mutant does not, however, progress beyond this point. Examination of histone modifications in c(2)M mutants confirmed that synapsis is blocked in mid-zygotene with a small number of euchromatin initiation sites. Based on the similarities between wild-type mid-zygotene and c(2)M mutants, it is suggested that synapsis initiates in a c(2)M-independent manner at a small number of specialized sites on the chromosomes, which include approximately six euchromatic sites and the centromeres, and that C(2)M is required for additional initiation sites typical of late zygotene (Tanneti, 2011).

There is a striking similarity between the number of euchromatic synapsis initiation sites (~6) during mid-zygotene and the number of crossovers in Drosophila females. In order to determine the relationship between SC initiation sites and double-strand break (DSB) formation, c(2)M mutant oocytes were stained for C(3)G and γ-H2AV. DSBs in a c(2)M mutant are usually associated with a patch of C(3)G staining (55/56 γ-H2AV foci were touching or overlapped a patch of C(3)G). This experiment was also performed in an okr mutant background (okr encodes the Drosophila homolog of Rad54) where the DSBs are not repaired and γ-H2AV staining accumulates, allowing all DSBs to be counted. Most of the γ-H2AV foci in okr c(2)M mutant germaria colocalized with a patch of C(3)G, suggesting that the initiation of SC and recombination usually occur within the same region in c(2)M mutants. Indeed, MEI-P22, a protein required for DSB formation, also colocalizes with the SC in c(2)M mutant oocytes. It should be noted that previous observations showed that DSB formation is partially dependent on the SC. Indeed, the number of γ-H2AV foci in the okr c(2)M double mutant in region 3 oocytes was reduced compared to a okr single mutant. Overall, these results suggest that the SC, or a factor which stimulates SC formation, promotes recruitment of proteins required for DSB formation (Tanneti, 2011).

To investigate whether there is a connection between early SC initiation events and meiotic recombination, double mutants with c(2)M were constructed. Unlike wild-type, where γ-H2AV foci are not observed until pachytene, the block in synapsis observed in c(2)M mutants allowed examination of the relationship between SC initiation and DSB formation. By double staining with CID, it was found that eliminating meiotic DSBs with a mei-W68 mutation did not prevent formation of either the centromere and euchromatic SC in a c(2)M mutant. The small decrease in the number of euchromatic SC sites in the c(2)M mei-W68 double mutant may indicate that the number of initiation sites is sensitive to DSB formation. Furthermore, SC initiation is not grossly affected by a reduction in crossing over (mei-218), an increase in crossing over (TM6), or a defect in DSB repair (okr). DSBs do not occur in the heterochromatin; thus, it is not surprising that centromere SC is independent of DSB formation. However, these results show that the initiation of euchromatic synapsis at mid zygotene does not depend on DSBs or crossovers (Tanneti, 2011).

Because DSBs or recombination are not required for synapsis in wild-type or c(2)M mutants, tests were performed to see whether structural components of the meiotic chromosomes regulate SC initiation. ORD is a meiosis-specific protein required for cohesion and crossover formation that may be a component of the SC lateral elements. Although previous studies have shown that ord mutant oocytes generate threads of C(3)G staining that resemble pachytene, the effect of ord on zygotene progression has not been previously examined (Tanneti, 2011).

Consistent with previous results, this study found that centromere clustering is defective and the association of SC proteins with the centromeres is disrupted in ord mutant oocytes. Furthermore, zygotene appeared abnormal; rather than observing centromeric and euchromatic SC initiation sites typical of mid-zygotene in early region 2a, it was found that many ord mutant pro-oocytes with C(3)G staining only around the nuclear DNA. Of the 108 pro-oocytes examined in five germaria, 36 (33%) had no nuclear C(3)G. The remaining pro-oocytes [72, (67%)] either had a number of C(3)G patches that was more typical of late zygotene, usually in region 2a, or were in pachytene. It is concluded that the centromeric and euchromatic synapsis sites typical of early and mid zygotene are absent in ord mutants, suggesting that, in the absence of ORD, synapsis does not initiate normally (Tanneti, 2011).

Because ord mutants do eventually form threads of SC, it was difficult to be sure that SC initiation was defective. To test whether ord has a role in mid-zygotene synapsis, tests were performed to see whether the euchromatic patches of C(3)G in a c(2)M mutant depend on ord. Even though both single mutants exhibit at least some SC formation, most of the C(3)G staining in the c(2)M ord double mutant surrounded the DNA and within the nucleus. This nonchromosomal C(3)G localization in the c(2)M ord double mutant was much more pronounced than in the ord single mutant. In addition, C(3)G-staining ring-like structures were observed similar to what has been reported in some c(3)G missense mutants. All the nonchromosomal C(3)G staining may be due to polycomplex formation. c(2)M ord double mutant pro-oocytes were identified by the prominent C(3)G around the DNA, and the number of C(3)G patches on the chromosomes was found to be drastically reduced compared to wild-type zygotene or either single mutant (Tanneti, 2011).

These results demonstrate that ord is required for the centromeric and euchromatic synapsis sites observed in c(2)M mutants. Conversely, C(2)M is required for the threadlike synapsis observed in ord mutants. The synergistic phenotype of the double mutant suggests that there are two types of synapsis initiation - one depends on ORD (early and mid-zygotene) and the other depends on C(2)M (late zygotene) - and that these are independent events. In the absence of both types of synapsis initiation, C(3)G cannot load onto the chromosomes and accumulates in polycomplexes (Tanneti, 2011).

Like other Kleisin family members, C(2)M has been shown to physically interact with the cohesin subunit SMC3 (Heidmann, 2004). To determine whether C(2)M localization depends on an interaction with cohesin, oocytes lacking SMC1 and SMC3 were examined. To examine oocytes lacking SMC3 (encoded by cap), the recently developed short hairpin RNA (shRNA) resource, which allows RNA interference (RNAi) knockdown of gene expression in the Drosophila female germline, was used. Both the chromosomal localization of C(3)G and C(2)M were absent when cap shRNA was expressed in the germline. Furthermore, SMC1 staining was eliminated, suggesting that the RNAi was effective at knocking out SMC3 function. Like the c(2)M ord double mutant, most C(3)G staining accumulated around the periphery of the DNA, suggesting that the function of SMC3 in synapsis occurs through at least two independent interactions with C(2)M and ORD. Unlike the c(2)M ord double mutant, however, it was not possible to distinguish the pro-oocytes from the nurse cells because C(3)G staining was evenly distributed among the cells in each germarium cyst. Importantly, oocyte selection was not perturbed because one cell in each cyst accumulated ORB protein, a cytoplasmic marker for the oocyte. Thus, the loss of SMC3 may have a more severe phenotype than the c(2)M ord double mutant (Tanneti, 2011).

These results were confirmed with the analysis of SMC1 mutant germline clones. As with cap RNAi, there was an absence of nuclear C(2)M and C(3)G threads in oocytes lacking SMC1, indicating a complete block in synapsis. Also similar to cap RNAi, the accumulation of ORB in one cell indicated that an oocyte was established. The only difference compared to cap RNAi was that there was much less C(3)G staining around the periphery of the DNA. It is not known whether this minor difference is due to the different methods (RNAi versus germline clone) or distinct functions of the two SMC proteins. Nevertheless, the results of these two experiments demonstrate that SMC1 and SMC3 are required for synapsis (Tanneti, 2011).

It is concluded that synapsis initiation during zygotene in Drosophila females occurs in three stages. In early zygotene, the centromeres are the first sites to accumulate the transverse filament protein C(3)G. Indeed, cohesion proteins SMC1, SMC3, and ORD are detected at the centromeres before meiotic prophase (prior to or during premeiotic S phase), which could explain why synapsis is first observed at the centromeres. Interestingly, the SC also forms first at the centromeres in budding yeast and depends on cohesion proteins. In mid-zygotene, synapsis initiates at a small number of euchromatic sites. These first two steps depend on the ORD protein. Finally, in late zygotene, synapsis initiates at a larger number of euchromatic sites. This stage requires C(2)M and appears to occur through a new set of initiation events rather than extending synapsis, or 'zipping up,' from the mid-zygotene initiation sites. Indeed, the synapsis initiation events in mid and late zygotene are independent and genetically separable, supporting a model where synapsis occurs through two independent waves of initiation events. In the absence of ORD, early and mid-zygotene synapsis events are skipped and the late zygotene initiation events occur with normal kinetics. This is not without consequence, however, because at the electron microscopy level, this synapsis is abnormal and tripartite SC is not visible. Both waves of synapsis initiation depend on the cohesin proteins SMC1 and SMC3, which may interact independently with C(2)M and ORD (Tanneti, 2011).

In addition to its role in centromere synapsis, ORD and the SMC proteins are required for the pairing and clustering of centromeres, whereas the SC components C(2)M or C(3)G are not. Thus, cohesion proteins may be able to function in a pairing role independent of DSBs, as Rec8 does in budding yeast for centromere coupling. It is suggested that the first euchromatic sites to initiate SC assembly in Drosophila are in regions where cohesion proteins are most abundant. This model is attractive because it provides a mechanism for SC initiation in the absence of DSBs. Interestingly, the number of euchromatic initiation sites in mid-zygotene or in c(2)M mutants approximates the number of crossovers in the genome. Not only do these mid-zygotene sites depend on ORD, but in ord mutants, crossing over is reduced to less than 10% of wild-type, even though DSBs occur normally. It is suggested that the reduction in crossing over in ord mutants is due to the absence of the synapsis initiation sites at mid-zygotene. Whether the synapsis initiation sites actually correspond to crossover sites awaits further study (Tanneti, 2011).

ORD may have a function similar to yeast Rec8 because it is required for synapsis at the centromeres and a subset of euchromatic sites. Interestingly, the findings with C(2)M, which is not an ortholog of Rec8, are also probably relevant to other species. Several recent studies have revealed Non-Rec8 Kleisin homologs in mouse and C. elegans (COH-3 and COH-). These parallels between the synapsis pathway in flies and that of organisms that depend on DSBs for synapsis could reflect the existence of a conserved underlying mechanism of synapsis. If synapsis initiation sites can be marked prior to DSB formation in a process involving cohesion proteins, and if proteins like Zip3 can be recruited in the absence of DSBs, as is true in C. elegans and likely in Drosophila, the timing of the DSB then becomes less of a determining factor in the process of synapsis (Tanneti, 2011).

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 ordnull 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 ordnull 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 ordnull 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 ID: 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 ID: 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 ID: 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 ID: 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 ID: 12062057

Goldstein, L. S. (1980). Mechanisms of chromosome orientation revealed by two meiotic mutants in Drosophila melanogaster. Chromosoma 78: 79-111. PubMed ID: 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 ID: 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 ID: 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 ID: 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 ID: 12593793

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

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

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

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

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

Page, S. L., and Hawley, R. S. (2004). The genetics and molecular biology of the synaptonemal complex. Annu. Rev. Cell Dev. Biol. 20: 525-558. PubMed ID: 15473851

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 ID: 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 ID: 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 ID: 12667442

Shintomi, K. and Hirano, T. (2007). How are cohesin rings opened and closed? Trends Biochem Sci. 32: 154-157. PubMed ID: 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 ID: 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 ID: 14563680

Tanneti, N. S., Landy, K., Joyce, E. F. and McKim, K. S. (2011). A pathway for synapsis initiation during zygotene in Drosophila oocytes. Curr. Biol. 21(21): 1852-7. PubMed ID: 22036181

Watts, F. Z., and Hoffmann, E. (2011). SUMO meets meiosis: An encounter at the synaptonemal complex: SUMO chains and sumoylated proteins suggest that heterogeneous and complex interactions lie at the centre of the synaptonemal complex. Bioessays 33: 529-537. PubMed ID: 21590786

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 ID: 15007062

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