Publications by authors named "Christa Heyting"

12 Publications

  • Page 1 of 1

Cytological analysis of interference in mouse meiosis.

Methods Mol Biol 2009 ;558:355-82

Molecular Genetics Group, Wageningen University and Research Center, Wageningen, The Netherlands.

In most eukaryotes, meiotic crossovers (COs) are non-randomly placed along the bivalents, such that the presence of a CO reduces the probability of additional COs nearby. This phenomenon, named CO interference, was originally defined genetically, but can also be analyzed cytologically by studying the chromosomal positions of protein complexes that are involved in CO formation, or by studying the positions of chiasmata. Here we focus on the cytological analysis of interference among protein complexes involved in meiotic recombination and CO formation in the mouse. During the pachytene stage of meiosis, these protein complexes can be visualized as immunofluorescent foci along synaptonemal complexes (SCs), which are linear protein structures that are formed along homologous chromosome pairs (bivalents) during meiotic prophase. We describe how to make cytological preparations that are suitable for the analysis of interference among these foci, and how to estimate the strength of interference among foci, using the gamma distribution as a mathematical model for focus/CO positioning.
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http://dx.doi.org/10.1007/978-1-60761-103-5_21DOI Listing
October 2009

Role of cleavage by separase of the Rec8 kleisin subunit of cohesin during mammalian meiosis I.

J Cell Sci 2009 Aug;122(Pt 15):2686-98

Research Institute of Molecular Pathology, A-1030 Vienna, Austria.

Proteolytic activity of separase is required for chiasma resolution during meiosis I in mouse oocytes. Rec8, the meiosis-specific alpha-kleisin subunit of cohesin, is a key target of separase in yeast. Is the equivalent protein also a target in mammals? We show here that separase cleaves mouse Rec8 at three positions in vitro but only when the latter is hyper-phosphorylated. Expression of a Rec8 variant (Rec8-N) that cannot be cleaved in vitro at these sites causes sterility in male mice. Their seminiferous tubules lack a normal complement of 2 C secondary spermatocytes and 1 C spermatids and contain instead a high proportion of cells with enlarged nuclei. Chromosome spreads reveal that Rec8-N expression has no effect in primary spermatocytes but produces secondary spermatocytes and spermatids with a 4 C DNA content, suggesting that the first and possibly also the second meiotic division is abolished. Expression of Rec8-N in oocytes causes chromosome segregation to be asynchronous and delays its completion by 2-3 hours during anaphase I, probably due to inefficient proteolysis of Rec8-N by separase. Despite this effect, chromosome segregation must be quite accurate as Rec8-N does not greatly reduce female fertility. Our data is consistent with the notion that Rec8 cleavage is important and probably crucial for the resolution of chiasmata in males and females.
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http://dx.doi.org/10.1242/jcs.035287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909317PMC
August 2009

Cytological analysis of MRE11 protein during early meiotic prophase I in Arabidopsis and tomato.

Chromosoma 2008 Jun 22;117(3):277-88. Epub 2008 Feb 22.

Department of Biology, Colorado State University, Fort Collins, CO 80523, USA.

Early recombination nodules (ENs) are multiprotein complexes that are thought to be involved in synapsis and recombination, but little is known about their components or how they may be involved in these events. In this study, we describe the cytological behavior of a possible EN component, MRE11, a protein that is important for the repair of the numerous, programmed deoxyribonucleic acid double-strand breaks (DSBs) that occur early in the meiotic prophase. By immunofluorescence, many MRE11 foci were associated with chromosomal axes during early prophase I in both wild-type Arabidopsis and tomato primary microsporocytes. Similar patterns of MRE11 foci were observed in two Arabidopsis mutants (Atspo11-1 and Atprd1) that are defective in DSB formation and synapsis. In tomato chromosomes, MRE11 foci were more common in distal euchromatin than in proximal heterochromatin, consistent with known EN patterns. However, electron microscopic immunogold localization demonstrated that only about 10% of ENs were labeled, and most MRE11 label was associated with synaptonemal complex components. Thus, in plants, MRE11 foci are not dependent on DSB formation, and most MRE11 foci do not correspond to ENs. More generally, our results show that the simple presence of large numbers of fluorescent foci associated with synapsing chromosomes is insufficient evidence to equate these foci with ENs.
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http://dx.doi.org/10.1007/s00412-007-0147-zDOI Listing
June 2008

The mismatch repair protein MLH1 marks a subset of strongly interfering crossovers in tomato.

Plant Cell 2007 Mar 2;19(3):862-76. Epub 2007 Mar 2.

Wageningen University and Research Centre, Molecular Genetics Group, NL-6703BD Wageningen, The Netherlands.

In most eukaryotes, the prospective chromosomal positions of meiotic crossovers are marked during meiotic prophase by protein complexes called late recombination nodules (LNs). In tomato (Solanum lycopersicum), a cytological recombination map has been constructed based on LN positions. We demonstrate that the mismatch repair protein MLH1 occurs in LNs. We determined the positions of MLH1 foci along the 12 tomato chromosome pairs (bivalents) during meiotic prophase and compared the map of MLH1 focus positions with that of LN positions. On all 12 bivalents, the number of MLH1 foci was approximately 70% of the number of LNs. Bivalents with zero MLH1 foci were rare, which argues against random failure of detecting MLH1 in the LNs. We inferred that there are two types of LNs, MLH1-positive and MLH1-negative LNs, and that each bivalent gets an obligate MLH1-positive LN. The two LN types are differently distributed along the bivalents. Furthermore, cytological interference among MLH1 foci was much stronger than interference among LNs, implying that MLH1 marks the positions of a subset of strongly interfering crossovers. Based on the distances between MLH1 foci or LNs, we propose that MLH1-positive and MLH1-negative LNs stem from the same population of weakly interfering precursors.
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http://dx.doi.org/10.1105/tpc.106.049106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1867368PMC
March 2007

Meiotic interference among MLH1 foci requires neither an intact axial element structure nor full synapsis.

J Cell Sci 2007 Mar 13;120(Pt 5):731-6. Epub 2007 Feb 13.

Wageningen University, Molecular Genetics group, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands.

During meiosis, homologous chromosomes (homologs) perform reciprocal exchanges (crossovers) at a high frequency. Crossovers display interference, i.e. their spacing is more even than would be expected if they were placed randomly along the chromosomes. Concomitantly with crossover formation, synaptonemal complexes (SCs) appear between homologs: each chromosome forms an axial structure, the axial element (AE); the AEs of homologs align, and numerous transverse filaments connect the AEs to form an SC. Both the AE and the SC have been implicated in the imposition of interference. We investigated whether intact AEs or SCs are required for crossover interference in the mouse, using a mutant lacking AE protein SYCP3, which displays structurally abnormal AEs and incomplete synapsis. We estimated the level of interference from the spacing of immunofluorescent MLH1 foci, which mark almost all crossover sites in the mouse, along the SCs. The levels of interference among MLH1 foci in wild-type and Sycp3(-/-) mice were comparable, implying that neither an intact AE structure nor full synapsis is required for wild-type levels of interference.
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http://dx.doi.org/10.1242/jcs.003186DOI Listing
March 2007

Two levels of interference in mouse meiotic recombination.

Proc Natl Acad Sci U S A 2006 Jun 9;103(25):9607-12. Epub 2006 Jun 9.

Molecular Genetics Group, Wageningen University and Research Centre, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands.

During meiosis, homologous chromosomes (homologs) undergo recombinational interactions, which can yield crossovers (COs) or noncrossovers. COs exhibit interference; they are more evenly spaced along the chromosomes than would be expected if they were placed randomly. The protein complexes involved in recombination can be visualized as immunofluorescent foci. We have analyzed the distribution of such foci along meiotic prophase chromosomes of the mouse to find out when interference is imposed and whether interference manifests itself at a constant level during meiosis. We observed strong interference among MLH1 foci, which mark CO positions in pachytene. Additionally, we detected substantial interference well before this point, in late zygotene, among MSH4 foci, and similarly, among replication protein A (RPA) foci. MSH4 foci and RPA foci both mark interhomolog recombinational interactions, most of which do not yield COs in the mouse. Furthermore, this zygotene interference did not depend on SYCP1, which is a transverse filament protein of mouse synaptonemal complexes. Interference is thus not specific to COs but may occur in other situations in which the spatial distribution of events has to be controlled. Differences between the distributions of MSH4/RPA foci and MLH1 foci along synaptonemal complexes might suggest that CO interference occurs in two successive steps.
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http://dx.doi.org/10.1073/pnas.0600418103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1475796PMC
June 2006

The diverse roles of transverse filaments of synaptonemal complexes in meiosis.

Chromosoma 2006 Jun 8;115(3):220-34. Epub 2006 Mar 8.

Botanical centre, Molecular genetics group, Wageningen University, Arboretumlaan 4, 6703 BD, Wageningen, The Netherlands.

In most eukaryotes, homologous chromosomes (homologs) are closely apposed during the prophase of the first meiotic division by a ladderlike proteinaceous structure, the synaptonemal complex (SC) [Fawcett, J Biophys Biochem Cytol 2:403-406, 1956; Moses, J Biophys Biochem Cytol 2:215-218, 1956]. SCs consist of two proteinaceous axes, which each support the two sister chromatids of one homolog, and numerous transverse filaments (TFs), which connect the two axes. Organisms that assemble SCs perform meiotic recombination in the context of these structures. Although much information has accumulated about the composition of SCs and the pathways of meiotic crossing over, several questions remain about the role of SCs in meiosis, in particular, about the role of the TFs. In this review, we focus on possible role(s) of TFs. The interest in TF functions received new impulses from the recent characterization of TF-deficient mutants in a number of species. Intriguingly, the phenotypes of these mutants are very different, and a variety of TF functions appear to be hidden behind a façade of morphological conservation. However, in all TF-deficient mutants a specific class of crossovers that display interference is affected. TFs appear to create suitable preconditions for the formation of these crossovers in most species, but are most likely not directly involved in the interference process itself. Furthermore, TFs are important for full-length homolog alignment.
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http://dx.doi.org/10.1007/s00412-006-0057-5DOI Listing
June 2006

Meiotic transverse filament proteins: essential for crossing over.

Authors:
Christa Heyting

Transgenic Res 2005 Oct;14(5):547-50

Molecular genetics Group, Wageningen University and Research Center, Arboretumlaan 4, 6703 Wageningen, BD, The Netherlands.

Meiosis is a specialized set of two nuclear divisions, meiosis I and II, by which a diploid cell produces four haploid daughters. After premeiotic DNA replication, homologous chromosomes pair and recombine, and then disjoin at meiosis I. Subsequently, at meiosis II, the sister chromatids of each chromosome segregate. In nearly all eukaryotes, meiotic chromosome pairing culminates in the formation of a ladder-like supramolecular protein structure, the synaptonemal complex (SC) (Page and Hawley, 2004). The rungs of the ladder are known as transverse filaments (TFs). Genes encoding TF proteins have been identified in a limited number of organisms, and their function has been studied by mutational analysis. Although TF proteins show little amino acid sequence conservation, their structure and function are largely conserved. In all analyzed species, TF proteins are required for meiotic reciprocal recombination (crossing over).
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http://dx.doi.org/10.1007/s11248-005-8925-yDOI Listing
October 2005

Mouse Sycp1 functions in synaptonemal complex assembly, meiotic recombination, and XY body formation.

Genes Dev 2005 Jun;19(11):1376-89

Department of Toxicogenetics, Leiden University Medical Centre, The Netherlands.

In meiotic prophase, synaptonemal complexes (SCs) closely appose homologous chromosomes (homologs) along their length. SCs are assembled from two axial elements (AEs), one along each homolog, which are connected by numerous transverse filaments (TFs). We disrupted the mouse gene encoding TF protein Sycp1 to analyze the role of TFs in meiotic chromosome behavior and recombination. Sycp1(-/-) mice are infertile, but otherwise healthy. Sycp1(-/-) spermatocytes form normal AEs, which align homologously, but do not synapse. Most Sycp1(-/-) spermatocytes arrest in pachynema, whereas a small proportion reaches diplonema, or, exceptionally, metaphase I. In leptotene Sycp1(-/-) spermatocytes, gammaH2AX (indicative of DNA damage, including double-strand breaks) appears normal. In pachynema, Sycp1(-/-) spermatocytes display a number of discrete gammaH2AX domains along each chromosome, whereas gammaH2AX disappears from autosomes in wild-type spermatocytes. RAD51/DMC1, RPA, and MSH4 foci (which mark early and intermediate steps in pairing/recombination) appear in similar numbers as in wild type, but do not all disappear, and MLH1 and MLH3 foci (which mark late steps in crossing over) are not formed. Crossovers were rare in metaphase I of Sycp1(-/-) mice. We propose that SYCP1 has a coordinating role, and ensures formation of crossovers. Unexpectedly, Sycp1(-/-) spermatocytes did not form XY bodies.
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http://dx.doi.org/10.1101/gad.329705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1142560PMC
June 2005

Cohesin SMC1 beta is required for meiotic chromosome dynamics, sister chromatid cohesion and DNA recombination.

Nat Cell Biol 2004 Jun 16;6(6):555-62. Epub 2004 May 16.

Center for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA.

Sister chromatid cohesion ensures the faithful segregation of chromosomes in mitosis and in both meiotic divisions. Meiosis-specific components of the cohesin complex, including the recently described SMC1 isoform SMC1 beta, were suggested to be required for meiotic sister chromatid cohesion and DNA recombination. Here we show that SMC1 beta-deficient mice of both sexes are sterile. Male meiosis is blocked in pachytene; female meiosis is highly error-prone but continues until metaphase II. Prophase axial elements (AEs) are markedly shortened, chromatin extends further from the AEs, chromosome synapsis is incomplete, and sister chromatid cohesion in chromosome arms and at centromeres is lost prematurely. In addition, crossover-associated recombination foci are absent or reduced, and meiosis-specific perinuclear telomere arrangements are impaired. Thus, SMC1 beta has a key role in meiotic cohesion, the assembly of AEs, synapsis, recombination, and chromosome movements.
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http://dx.doi.org/10.1038/ncb1135DOI Listing
June 2004

Meiotic cohesin REC8 marks the axial elements of rat synaptonemal complexes before cohesins SMC1beta and SMC3.

J Cell Biol 2003 Mar;160(5):657-70

Molecular Genetics Group, Botanical Center, Wageningen University, Arboretumlaan 4, 6703 BD Wageningen, Netherlands.

In meiotic prophase, the sister chromatids of each chromosome develop a common axial element (AE) that is integrated into the synaptonemal complex (SC). We analyzed the incorporation of sister chromatid cohesion proteins (cohesins) and other AE components into AEs. Meiotic cohesin REC8 appeared shortly before premeiotic S phase in the nucleus and formed AE-like structures (REC8-AEs) from premeiotic S phase on. Subsequently, meiotic cohesin SMC1beta, cohesin SMC3, and AE proteins SCP2 and SCP3 formed dots along REC8-AEs, which extended and fused until they lined REC8-AEs along their length. In metaphase I, SMC1beta, SMC3, SCP2, and SCP3 disappeared from the chromosome arms and accumulated around the centromeres, where they stayed until anaphase II. In striking contrast, REC8 persisted along the chromosome arms until anaphase I and near the centromeres until anaphase II. We propose that REC8 provides a basis for AE formation and that the first steps in AE assembly do not require SMC1beta, SMC3, SCP2, and SCP3. Furthermore, SMC1beta, SMC3, SCP2, and SCP3 cannot provide arm cohesion during metaphase I. We propose that REC8 then provides cohesion. RAD51 and/or DMC1 coimmunoprecipitates with REC8, suggesting that REC8 may also provide a basis for assembly of recombination complexes.
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http://dx.doi.org/10.1083/jcb.200212080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2173354PMC
March 2003

The pairing of X and Y chromosomes during meiotic prophase in the marsupial species Thylamys elegans is maintained by a dense plate developed from their axial elements.

J Cell Sci 2003 Feb;116(Pt 3):551-60

Programa de Genética Humana, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile.

Unlike eutherian males, pairing of the sex chromosomes in marsupial males during the first meiotic prophase is not mediated by a synaptonemal complex. Instead, a specific structure, the dense plate, develops during pachytene between the sex chromosomes. We have investigated the development and structural nature of this asynaptic association in males of the marsupial species Thylamys elegans by means of immunolabelling and electron microscopy techniques. Our results show that the behaviour of male marsupial sex chromosomes during first meiotic prophase is complex, involving modifications of their structure and/or composition. Pairing of the sex chromosomes and formation of the dense plate take place in mid pachytene, paralleling morphological changes in the sex chromosomal axial elements. Components of the central element of the synaptonemal complex were not found in the sex body, in agreement with ultrastructural studies that reported the absence of a canonical tripartite synaptonemal complex between male marsupial sex chromosomes. Interestingly, the dense plate is labelled with antibodies against the SCP3 protein of the lateral elements of the synaptonemal complex. Moreover, as sex chromosome axial elements decrease in mass throughout mid-late pachytene, the dense plate increases, suggesting that material moves from the axial elements to the dense plate. Additionally, both sex chromosome axial elements and the dense plate have proteins that are specifically phosphorylated, as revealed by their labelling with the MPM-2 antibody, indicating that they undergo a chromosome-specific regulation process throughout first meiotic prophase. We propose that the unique modifications of the composition and structure of the axial elements of the sex chromosomes in meiotic prophase may result in the prescription of synaptonemal complex formation between male marsupial sex chromosomes, where the dense plate is an extension of the axial elements of sex chromosomes. This replaces synapsis to maintain X and Y association during first meiotic prophase.
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http://dx.doi.org/10.1242/jcs.00252DOI Listing
February 2003