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5th European Meiosis Meeting
Canterbury U.K., April 3-6 2001
ABSTRACTS
C2.1 - Interference and non-interference recombination pathways in yeast
Frank Stahl and Jette Foss, University of Oregon
msh4-group mutations, which eliminate interference while reducing crossing over but not
conversion, suggest that yeast has an interference and a non-interference recombination
pathway (1) and that interference results from programmed resolution of intermediates.
At ARG4, on VIII where interference is often strong (2), cutting of Holliday junctions to
produce crossovers is biased, and noncrossovers may arise without any junction cutting
(3), suggesting that DSBR intermediates in the interference pathway are unligated.
Resolution, by unwinding, yields noncrossovers, except in intermediates stabilized by
Msh4-group proteins, where the unligated ends direct cutting of equipolar strands. This
implies that the canonical resolutions, which constitute a significant minority among
DSBR products at HIS4, on III where interference is often weak (2), are derived from
ligated intermediates, and that these products lack interference. These implications
harmonize with observations that in msh4-group mutants regions with weaker
interference show a smaller reduction in crossing over, reflecting a greater residue of
non-interfering crossovers (4). We suppose that interference occurs when clusters of
unligated DSBR intermediates (early nodules), gathered to form late nodules, allow only
one member of each cluster to bind Msh4-group proteins (5). The clustering of DSBR
sites as a prerequisite for crossover interference is supported by the observation that zip1
mutants retain nodule interference while losing crossover interference (6).
(1) Hasenkampf 1996; Zalevsky et al. 1999. (2) King and Mortimer 1991. (3) Gilbertson
and Stahl 1996. (4) Tung and Roeder 1998; Khazanehdari and Borts 2000. (5) Stahl
1993. (6) S. Roeder pers. com.
C2.2 - B-type cyclins CLB5 and CLB6 control the initiation of recombination and
synaptonemal complex formation in yeast meiosis
Kathleen N. Smith*, Alexandra Penkner‡, Kunihiro Ohta§, Franz Klein‡ and Alain
Nicolas*
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*Institut Curie, Section de Recherche, CNRS UMR 144, 26, rue d'Ulm, 75248 Paris Cedex 05, France; ‡
Department of Cytology and Genetics, Institute of Botany, University of Vienna, Rennweg 14, A-1030
Vienna, Austria; §Genetic Dynamics Research Unit-Laboratory, The Institute of Physical and Chemical
Research (RIKEN), Wako, Saitama 351-01, Japan
Meiotic DNA replication precedes the initiation of recombination by programmed Spo11dependent DNA double-strand breaks (DSBs). Recent reports that meiosis-specific
cohesion is established during meiotic S phase and that the length of S phase is modified
by recombination factors (the DSB nuclease Spo11 and the sister chromatid cohesin
Rec8) raise the possibility that replication plays a fundamental role for the recombination
process. To address how replication influences the initiation of recombination we have
used mutations in the B-type cyclin genes CLB5 and CLB6 which specifically prevent
premeiotic replication in the yeast Saccharomyces cerevisiae but which do not trigger the
replication-dependent checkpoint MEC1 (Genes Dev. 12:2698-2710; Science 281:18541857), thereby allowing cells to progress through meiosis. We find that clb5 and clb5
clb6 but not clb6 mutants are defective in DSB induction and prior associated changes in
chromatin accessibility, heteroallelic recombination, and synaptonemal complex
formation and that the severity of these phenotypes in each mutant parallels the extent of
impairment of replication. Transcription of numerous early meiosis-specific genes
required for DSB formation, recombination, and SC development takes place in clb5 clb6
cells, indicating that these mutants are capable of executing the meiotic transcriptional
program. This assemblage of phenotypes reveals roles for CLB5 and CLB6 not only in
DNA replication but also in other key events of meiotic prophase. Links between the
function of CLB5 and CLB6 in activating meiotic DNA replication and their effects on
subsequent events will be discussed.
C2.3 - Starting and Finishing Meiotic Recombination
M. Lichten, T. Allers, V. Borde, R. Shroff and T.-C. Wu, NCI, Bethesda, MD (USA)
Starting: Double-strand DNA breaks (DSBs) initiate meiotic recombination. Recent
work has shown that the following contribute to determining where DSBs form: DNA
replication: Deleting active replication origins from an arm of chromosome III causes a
local delay of both replication and DSB formation in wild-type cells, and a severe loss of
breaks in mutants that have DSB processing defects. We conclude that replication is
needed to potentiate DSB formation in a chromosome segment-autonomous manner.
Centromeres--Deleting a centromere and inserting it elsewhere on a chromosome
increases DSBs in sequences adjacent to the old location and decreases DSBs near the
new centromere. Thus, yeast centromeres actively repress DSBs in their vicinity.
Centromere-associated factors responsible for DSB repression are being identified.
Finishing: The double-strand break-repair model (DSBR) of meiotic recombination
postulates a double Holliday junction intermediate that contains heteroduplex DNA, and
that is resolved to form either noncrossover or crossover recombinants. Contrary to this
expectation, we find that noncrossover and crossover recombinants are formed by
different mechanisms. In particular, we suggest that noncrossover recombinants form
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relatively early in meiosis I prophase and do not involve long-lived Holliday junctioncontaining intermediates. By contrast, crossovers are formed via long-lived intermediates
that are not resolved until the end of pachytene. These intermediates can contain
heteroduplex DNA, but some display features inconsistent with the DSBR model.
Alternative mechanisms for meiotic recombination will be presented.
C2.4 - Counting crossovers yeast: One two or three pathways?
M .F. F. Abdullah, K. Khazanehdari and R H Borts, Genetics, University of Leicester UK
The “mismatch repair” proteins Mlh1p, Mlh3p, Mlh2p, Msh4p, Msh5p, and Exo1p have
non-equivalent roles in recombination. Careful analysis of crossing-over and viability
patterns for mlh1, mlh3, msh4 and exo1 mutants in isogenic strains indicates different
effects for the loss of these proteins. Deletion of Exo1p or Mlh3p leads to a decrease in
crossing of a similar magnitude to that of Msh4p for Chromosome III intervals while the
effect of msh4 is greater on Chromosome VII. All have a greater effect than mlh1. Levels
of non-disjunction and the poorer viability in msh4 than exo1 or mlh3 could be explained
by loss of interference in msh4 but not exo1 or mlh3. Consistent with this we have
detected interference in exo1 strains. Because double mutants still display substantial
residual crossing over, we suggest that there is yet another minor crossover pathway that
may be used. Data consistent with this hypothesis comes from studying deletion of
Mlh2p. Although alone mlh2 has no effect on crossing over, double mutants between
mlh3 and mlh2 as well as msh4and mlh2 have substantially more recombination than the
single mutants. We hypothesised that Mlh2p blocks recombination intermediates
(perhaps only formed under mutant conditions) from going down a minor pathway.
Thus, in the absence of Mlh2p some recombination intermediates in msh4 or mlh3
strains can be rescued by this pathway. Interestingly, Interestingly, crossing over in a
triple mutant exo1mlh2 msh4 resembles mlh2 msh4 double indicating that Exo1p is not
essential for rescue.
C2.5 - Structural features of recombination complexes in S. cerevisiae
Douglas Bishop1, Miki Shinohara1,2, Jeremy Grushcow1, Heidi Olivares1, Caroline
Sham1, Edgar Trelles-Sticken3, Akira Shinohara1,2 and Harry Scherthan3
1 Department of Radiation and Cellular Oncology, University of Chicago, Chicago IL. 60637, USA, 2
Department of Biology, Osaka University, Toyonaka, Osaka 560-0043, Japan, 3 Univ. Kaiserslautern, D67663 Kaiserslautern, Germany
Two eukaryotic homologs of bacterial RecA, Rad51 and Dmc1, cooperate during
recombination to promote the conversion of DNA double strand breaks (DSBs) to
homologous joint molecules (JMs). During this process, the two proteins colocalize in
immunostaining foci. Colocalization depends on Tid1(Rdh54), a protein that binds both
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Rad51 and Dmc1. The observed colocalization is not precise. Foci are often partially
overlapping or arranged in closely-spaced pairs suggesting a Rad51 oligomer and a Dmc1
oligomer assemble at adjacent sites. We are currently testing the hypothesis that Rad51
assembles on one of the two ends created by a DSB while Dmc1 assembles on the other.
Like TID1, RAD17, a checkpoint control gene, influences recombination during the
conversion of DSBs to JMs. Genetic studies suggest that RAD17 helps the two DNA ends
created by a DSB recombine with the same locus. We are currently exploring the
relationship between the defects observed in rad17 to those observed in tid1 mutants.
Both mutants are defective in crossover interference providing further evidence that
crossover control is imposed while recombinase acts on DSBs. We speculate that proper
coordination of partner DNA ends during strand invasion is critical to regulating the
distribution of crossover recombinants.
C2.6 - Topological analysis of proteins involved in meiotic double-strand break
initiation and repair
Silvia Prieler, Ivana Knezic, Alexandra Penkner and Franz Klein, Institute of Botany,
Department of Cytology and Genetics, Rennweg 14, A-1030 Vienna, Austria
In yeast, just like in mice and humans, meiotic chromosomes interact with their homologs
through the repair of DNA-double strand breaks (DSBs), created by a highly regulated,
complex mechanism. This mechanism requires the conserved topoisomerase II relative,
Spo11, which catalyzes DSBs at a number of randomly activated hotspot sites. We have
earlier identified a gene COM1(SAE2), which is essential for repair of these meiotic
DSBs. Absence of Com1 leads to accumulation of unresected breaks with Spo11
covalently bound to the 5`-ends. As a consequence, pairing and synapsis are reduced,
nuclei become fragmented, chromosomes missegregate and the cells die. However, we
show that Com1 is also expressed during the vegetative cell cycle, suggesting a role in
vegetative DNA repair.
We have characterized Com1 regarding its expression, its modifications and its
localization in the cell and on chromosomes. Roughly half the protein is phosphorylated
at any time. In addition, a slower migrating form of Com1p arises parallel to the
appearance of meiotic DSBs, suggesting a possible mechanism of activation. Knocking
out components of the DNA damage checkpoint did not affect this modification. In situ
staining reveals that Com1 strictly resides in the nucleus of meiotic and mitotic cells. On
spreads Com1 localizes to foci on the chromatin during early stages of meiosis.
We have also analyzed Spo11. Our cytological analysis of Spo11 was
complemented by CHIP experiments (chromatin immuno precipitation) which allowed us
to determine the timing of covalent and non-covalent interaction of Spo11 with the DNA
at a recombination hotspot in vivo in meiosis. Simultaneous analysis of hotspot binding,
DSB formation, and in situ localization of Spo11 on chromosomes allow the correlation
between cytologically visible structures and molecular events. We find that maximal
Spo11 expression occurs after DSB formation. Moreover, Spo11 binds to chromosomes
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independent of, during and after the appearance of DSBs, suggesting a new and so far
unexpected role of Spo11 after DSB formation.
C2.7 - Analysis of recombination in wild-type meiosis and mlh1/3 and msh4/5
mutants: Implications for crossover control
Neil Hunter and Nancy Kleckner, Molecular and Cellular Biology, Harvard University, Cambridge,
MA02138, USA
Meiotic recombination is initiated by double-strand-breaks (DSBs) which convert to
double Holliday junctions (dHJs) and thence to products. We have now identified a new
DNA intermediate, single-end invasions (SEIs), which are the earliest detectable strandexchange intermediates between homologs. Our analysis confirms that DSBs occur
during leptotene and further shows: (i) the DSB to SEI transition is concomitant with
zygotene; (ii) SEIs emerge at the onset of pachytene; (iii) dHJs emerge at mid-pachytene
(previously correlated with pachytene DNA synthesis and crossover-specific
recombination nodules, LNs); (iv) crossovers appear just after pachytene. These results
imply that crossover control occurs prior to or during formation of SEIs. Implications for
two phenomena, two-phase synapsis and synaptic adjustment, will be discussed.
Homologs of DNA mismatch-repair proteins are required for meiotic
recombination. In yeast, heterodimers of Msh4+5, and Mlh1+3 function along a common
pathway to promote crossing-over. We show that msh4/5 cells lack crossover
interference and exhibit early DNA recombination defects: the DSBs to SEI transition is
severely delayed. Thus, events important for normal crossover control occur at/before
this transition, consistent with the conclusions made above. In sharp contrast, mlh1/3
mutants exhibit robust crossover interference, no DSB defect and little/no defect in
progression through later DNA stages, but nonetheless show significantly reduced
crossing-over. mlh1/3, therefore, define a new class of meiotic mutant in which crossingover is reduced but crossover control is unaltered. Since Mlh1 immunostaining foci
appear at mid-pachytene, in correlation with LNs, mlh1/3 cells appear to be defective in
implementing exchange at recombinational interactions previously designated to mature
as crossovers.
C2.8 - The role MMS4 in meiosis
N. M. Hollingsworth and T. de los Santos, Biochemistry and Cell Biology, SUNY Stony Brook;
J. Loidl, Cytology and Genetics, University of Vienna
The MMS4 gene of S. cerevisiae confers resistance in vegetative cells to alkylating agents
such as MMS, but is dispensable for repair of UV- and X-ray-induced damage. mms4
diploids sporulate poorly. We isolated MMS4 as a MEK1-interacting protein in a two-
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hybrid screen, suggesting that the mms4 sporulation defect may be due to a requirement
for MMS4 in meiotic recombination. Consistent with this idea, mms4 mutants exhibit a
2-3 fold decrease in interhomolog crossing over and spore viability, with little effect on
gene conversion. While these phenotypes are similar to those of a number of meiotic
mutants, including zip1 and msh5, mms4 differs in that the spore inviability is not due to
chromosome missegregation. Instead, we propose that aberrant recombination occurring
in the absence of MMS4 triggers the meiotic recombination checkpoint. At 33o, SK1
strains deleted for MMS4 arrest primarily at the mononucleate stage. The mms4 meiotic
progression defect can be rescued either by preventing the formation of meiotic double
strand breaks (DSBs), or by deletion of genes required for the meiotic recombination
checkpoint (PCH2, MEK1 and RED1). In spo13 diploids, red1, but not mek1, is epistatic
to mms4 for sporulation and spore viability, indicating that recombination occurring in
the absence of RED1 is not affected by MMS4. Chromosome synapsis is delayed and
incomplete in mms4 mutants. DSBs and crossover products are formed, but some DSBs
persist. These observations indicate that MMS4 plays a key role in meiotic
recombination, perhaps in the processing or resolution of recombination intermediates.
C2.9 - The C. elegans rad-51 gene is active in several pathways in meiosis and in
somatic tissues
A. La Volpe and C. Rinaldo, IIGB-CNR Naples, Italy
The recombination pathway and enzymology have been conserved throughout evolution,
for example the eukaryotic gene RAD51, first discovered in Saccharomyces cerevisiae
and found conserved in other fungi, plants and animals, is the structural and functional
homolog of RecA. Due to the extreme variation in cellular organization and
environmental stimuli in highly diverging organisms, while conserved molecules and
pathways are used, similar genes may adopt different regulation and interactions in
evolutionary distant species. Furthermore, elimination of conserved molecules or
interference on conserved pathways may lead to different results in different organisms
(Dernburg, 1998; Zalevsky, 1999; Zetka, 1999).
Genetic and biochemical studies on different model systems will, hopefully,
provide important contribution to the understanding of the crucial features and
fundamental components of the recombination pathways and elucidating how, in
individual species, evolution has led to the adaptation of molecules and functions to the
context of different environmental stimuli and of diverged cellular organization.
Among eukaryotes C. elegans is the only organism known so far that conserves a single
recA like gene, the homolog of RAD51 (Rinaldo, 1998; Takanami, 1998). In particular
the meiosis specific DMC1 gene, very similar to RAD51, that is present in fungi, plants
and mammals, is absent in C. elegans.
We will report a detailed analysis of the rad-51 severe loss of function phenotype
obtained by RNA- interference in different genetic backgrounds, trying to dissect the
various pathways in which this gene is involved in meiosis as well as in soma.
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C2.10 – Recombination proteins localize at the telomeres of mouse meiotic
chromosomes
M. Tarsounas, Imperial Cancer Research Fund, South Mimms, UK
C2.11 - Patterns of meiotic recombination in the human female
C. Tease, G. Hartshorne and M. Hultén, Biological Sciences, University of Warwick, U.K
Meiotic recombination is essential for the correct segregation of homologous
chromosomes in human germ cells. Variations in the patterns of maternal recombination
may be an important risk factor for meiotic chromosome nondisjunction. We have used a
recently developed method that for the first time allows direct investigation of
recombination patterns in human oocytes. This approach exploits the remarkable
behaviour of a DNA mismatch repair protein (MLH1) which forms discrete foci along
paired homologous chromosomes in germ cells at prophase I of meiosis. These foci
mirror the numbers and distributions of meiotic crossovers. Female meiosis initiates
during fetal development in humans. We have found that MLH1 foci first appear at early
zygotene and persist to diplotene in fetal oocytes. A mean of 70.3 (+/- 10.5) foci was
present in pachytene cells (n = 95) with considerable inter-cell variation (range 48 – 102).
This average of 70 foci (= 70 crossovers) per cell equates to a genetic length of 3500 cM.
Selected chromosome pairs were identified using FISH with chromosome-specific DNA
probes to allow analysis of the numbers and locations of their foci. Initial observations
yielded means of 2.5 (= 1250 cM) and 1.3 (= 650 cM) foci for chromosomes 13 and 21
respectively. Adjacent foci were almost invariably separated by considerable distances.
Foci rarely formed adjacent to the telomeres but generally were located interstitially.
Immunocytogenetical analysis produces unique information on inter- and intra-individual
variations in patterns of maternal recombination, which will be invaluable to our
understanding of the aetiology of aneuploidy.
C2.12 - Localization of Zip2 provides new insight into models of genetic interference
Jennifer Fung, Michael Odell and G. Shirleen Roeder, MCDB Dept. Yale University, New
Haven, CT 06520
In S. cerevisiae, the Zip2 protein promotes the initiation of synapsis between homologous
chromosomes during meiosis. Zip2 localizes to chromosomes in approximately 60
distinct foci per nucleus corresponding to sites of synapsis initiation. What determines
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the distribution of these sites? To address this question, the distribution of Zip2 foci on
pachytene chromosomes was determined cytologically. Tandem lacO repeats were
inserted at one end of chromosome XV in a strain expressing a GFP-LacI fusion protein
that binds to the lacO sequence. By measuring distances between Zip2 foci and the lacO
repeats, a cumulative distribution of Zip2 foci was obtained for each tagged chromosome.
The results indicate that synapsis initiation can occur anywhere along the chromosome.
Interestingly, Zip2 foci do display interference since the observed frequency of
Zip2 foci in adjacent chromosome intervals is 3- fold less than the frequency expected if
Zip2 foci are completely independent. In mutants showing reduced or abolished genetic
interference (zip1, msh4, tam1, tid1), interference of Zip2 foci remains unchanged. One
model for interference that could account for this observation is that interference is set up
in two steps. The first step requires that synapsis initiation sites be placed along the
chromosome in a pattern showing interference. Eventually, these sites will mark sites of
crossover. A second layer of interference results from exclusion of additional crossovers
from regions of Zip1 polymerization that originate at the synapsis initiation sites.
C2.13 - Introgression mapping reveals a 1:1 correspondence between chiasma
frequency and genetic distance and the relationship between physical and genetic
distance
Julie King 1, Huw M Thomas 1, Ian P Armstead 1, Neil R Jones 2, Michael J Kearsey 3,
Luned A Roberts 1, Gareth W Morgan 1 and Ian P King 1
1 Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion, SY23
3EB, Wales, UK. 2 Institute of Biological Sciences, University of Wales Aberystwyth, Ceredigion, SY23
3DA, Wales, UK. 3 School of Biosciences, The University of Birmingham, Birmingham B15 2TT, UK
The generation of genetic linkage maps has been one of the major focuses for geneticists
working on large genome plant and animal species. The ultimate aim of genetic linkage
maps is to facilitate the location of genes responsible for the control of agronomically and
scientifically important traits. DNA markers closely linked to a gene of interest are an
important tool for indirect selection in plant breeding programmes and in addition,
provide a springboard from which genes can be isolated via map based cloning strategies
such as chromosome walking, chromosome landing and chromosome jumping. However,
the results obtained from genetic mapping have raised a number of key questions of
fundamental importance regarding meiosis: 1) what is the relationship between chiasma
frequency and the size of genetic linkage maps, and 2) how does genetic distance relate to
physical distance from one part of the genome to another. Our research on the grasses has
revealed that a) there is a 1:1 correspondence between chiasma frequency and genetic
distance b) there are hotspots of recombination in the distal 12-18% of the chromosome
arms c) recombination close to and between the centromere and the NOR is suppressed d)
coding regions appear to be distributed along the whole length of the chromosome
including the centromere and NOR.
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C2.14 - Pairing, recombination, crossover control and synapsis
N. Kleckner, V. Boerner, J. Dekker, N. Hunter, Y. Blat and R. Protacio, Harvard University,
USA
I. Combinatorial determination of meiotic chromosome organization and function by
loops/axis and R/G-bands. Yeast meiotic chromosomal protein Red1 promotes DSB
formation and then modulates DSB development via RecA homolog Dmc1. Microarray
analysis reveals that Red1 binds all along the chromosome III axis, more abundantly in
R-bands than in a G-band. DSBs and Dmc1 occur in chromatin loop sequences, not
along the axis, but also are more abundant in R-bands than in a G-band. A red1∆
reduces both DSBs and Dmc loading. DSBs are reduced uniformly all along the
chromosome; Dmc1 loading is reduced differentially in R-bands. Thus: (1) Meiotic
recombination involves a tethered loop/axis collaboration: pre-DSB recombinosomes
form in chromatin loops, then migrate to chromosome axes, where DSBs occur; (2) Rbands are hyper-active for recombination initiation, independent of Red1, and are
particularly dependent upon Red1 for post-DSB development.
II. Interhomolog interactions via imposition and relief of stress. Chromosomes
undergo multiple sequential cycles of chromatin expansion and contraction
(“chromosome breathing”). Because expansion is constrained in various ways, breathing
results in corresponding cycles of imposition and relief of stress within, between and
along chromosomes. Meiotic prophase comprises three such cycles: leptotene/zygoteneearly pachytene; mid-pachytene/late pachytene; diffuse stage/diplotene. Meiotic
interhomolog interactions can be understood as a series of stress-promoted transitions,
each governed by the same basic principles. Overall, interhomolog interactions are
governed by mechanically interdependent effects of stresses imposed along the chromatin
fiber segments involved in recombination and along the chromosome axes.
C2.15 - Pairing and synapsis
D. Zickler, S. Tesse and A. Storlazzi, Université Paris-Sud, Orsay, France
Spo76p of Sordaria macrospora belongs to a highly conserved family implicated in
sister-chromatid cohesion and DNA repair. It localizes on chromosomes at all stages,
except from late prophase to telophase of mitosis and meiosis, and is involved in several
aspects of both programs (1). Spo76p is also required for meiotic interhomolog
recombination and its Aspergillus ortholog BIMD is required for mitotic interhomolog
recombination, but not for intrachromosomal recombination. Moreover, BIMD is a
negative regulator of normal cell cycle progression, suggesting that defects in
chromosome structure and/or sister cohesion can effect cell cycle progression (2).
We have carried out saturation genetic identification of mutations that suppress
the sister-chromatid cohesion defect of spo76-1. Seven "ASY" genes were identified, five
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of which are represented by 5-10 alleles. All but one mutant are defective in chromosome
synapsis and in synaptonemal complex formation. All are defective in meiotic
interhomolog recombination. All form axial elements and the availability of Spo76-GFP
has made possible the monitoring of their chromosome axis paths (by confocal and EM)
during the earliest stages of prophase. Homolog recognition, interlockings and bouquet
formation will be discussed in comparison with the wild type situation. The temporal
pattern of H3 phosphorylation was analyzed in parallel.
Mutations in the mitochondrial citrate synthase gene (cit1) reveal that the meiotic
diffuse stage is likely a metabolic checkpoint for meiotic completion (3). All 16 cit1
mutations impair meiotic progression after synapsis and most (including the null allele),
slow down both pairing and synapsis.
(1) van Heemst D., James F., Pöggeler S., Berteaux-L V., Zickler D. 1999. Cell, 98: 261271.
(2) van Heemst D., Kafer E., John T., Heyting C., van Aalderen M., Zickler D. 2001.
PNAS in press
(3) Ruprich-Robert G., Zickler D., Berteaux-L V., Picard M. in preparation
C2.16 - Functional dissection of in vivo interchromosome association in S. cerevisiae
Luis Aragon-Alcaide* and Alexander Strunnikov2
*MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Campus, Du Cane
Road, London W12 0NN ([email protected]), 2. Unit of Chromosome Structure and Function,
NICHD, NIH, 18T Library Drive, 106, Bethesda, Maryland 20892-5430, USA ([email protected])
The recognition of nucleic acid sequence homology within the nucleus has an impact on
seemingly disparate areas of nuclear function, from proper gamete formation during
meiosis to the epigenetic control of gene expression. Here, we investigate DNA to DNA
interactions using a non-invasive technique to tag and visualise DNA sequences in
vegetative and meiotic live cells. During meiosis the allelic position of the two
interacting-DNA tags play an imposing role, thus tag association depends on flanking
sequences. On the other hand, mitotic associations are based on the DNA sequence
homology of the tags, their genomic position and thus flanking regions have limited
effect on the interactions. This novel flanking-independent interactions, termed transassociation may underlie epigenetic phenomena.
C2.17 - Structure/function analysis of S. cerevisiae Spo11
PC Varoutas, C Mézard, JP Pin, A Nicolas and B de Massy
The S. cerevisiae Spo11 protein is thought to catalyze the formation of double-strand
breaks that initiate meiotic recombination based on three observations : 1) DSB breaks
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are not formed in spo11 null mutant, 2) Spo11 shares homology with the family of
TopoVI (DNA topoisomerase VI) catalytic subunits, 3) Spo11 is covalently attached to
the 5‘ ends of DNA in rad50S diploids. We have generated 16 point mutants of Spo11, at
conserved residues of the protein and analyzed their phenotypes according to various
meiotic assays. The positions of these mutations within Spo11 structure are predicted
from the 3D model structure we have generated based on the alignment between Spo11
and M. jannashii TopoVIA. This analysis allows us to define several critical residues in
the two domains of the protein, and also identifies wild-type alleles with substitutions at
conserved residues. In all cases, DSB formation is correlated with recombination
frequencies assayed by return to growth experiments. In addition, several new and
interesting observations come out from this analysis : 1) the identification of a partial loss
of function allele, 2) the co-dominant phenotype of some null mutants, 3) the unexpected
levels and distributions of viability in diploids with reduced spo11 activity.
C2.18 - Wild–type Spo11p is required for normal repair of double strand breaks
during meiosis
A.S.H. Goldman, M. Neale and M. Ramachandran, Molecular Biology and Biotechnology,
University of Sheffield, England
We have constructed SPO11 and spo11–y135f strains with an arg4 allele containing the
cutsite for the meiosis specific vde endonuclease and an arg4-bgl allele which acts as a
donor. Repair of the vde double strand break (DSB) can be via either inter–chromosomal
gene conversion or intra–chromosomal single strand annealing (SSA), using flanking
repeats of the URA3 gene. Genetic analysis in return to growth experiments reveals
reduced gene conversion frequencies when only mutant Spo11p is available. Analysis of
meiotic DNA supports this and indicates that repair is more often by SSA in the mutant
strain. We obtained similar results in strains wildtype for Spo11p but deficient for the
synaptic protein Hop1p, suggesting that the altered repair pattern in spo11-y125f strains
could be due to a chromosome pairing and/or synapsis fault. When the experiments are
repeated with the arg4 inserts dispersed on chromosomes V and III, the frequency of gene
conversion is still reduced in a spo11-y135f strain compared to a wildtype strain. The
ectopic data suggests that pairing status of the recombining chromosomes per se is not the
only factor influencing the proportion of gene conversions to SSA events in our assay.
One possibility is that bringing sequences close enough to recombine during meiosis is
dependent on normal chromosome pairing and/or synapsis in the cell as a whole, even
when the sequences are on heterologous chromosomes. It is also possible that wildtype
Spo11p is required, more directly, for normal regulation of meiotic DSB repair.
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C2.19 - Meiotic double-strand breaks and recombination are required for synapsis
in the mouse and the Spo11 protein is found on homologously synapsed
chromosomes
Peter J. Romanienko, Galina V. Petukhova, April L. Anderson and R. Daniel CameriniOtero, Genetics and Biochemistry Branch, NIDDK, National Institutes of Health Bethesda, MD 20892,
USA
The Spo11 protein initiates meiotic recombination by generating DNA double-strand
breaks (DSBs) and is required for meiotic synapsis in S. cerevisiae. Surprisingly, in C.
elegans and D. melanogaster Spo11 homologues are dispensable for synapsis yet
required for meiotic recombination. Disruption of mouse Spo11 results in infertility.
Spermatocytes arrest prior to pachytene with little or no synapsis and undergo apoptosis.
We did not detect Rad51/Dmc1 foci in meiotic chromosome spreads, indicating DSBs are
not formed. Cisplatin-induced DSBs restored Rad51/Dmc1 foci and promoted synapsis.
Spo11 localizes to discrete foci during leptotene, the stage when DSBs are generated, and
to homologously synapsed chromosomes at later stages. Other mouse mutants that arrest
during meiotic prophase (Atm -/-, Dmc1 -/-, mei1, Morc -/-) showed altered Spo11
protein localization and expression. The predominant form of Spo11 (Spo11-α) does not
contain exon 2, but in ATM, Dmc1 and Mei1 mutants (all of which arrest in early
prophase I) the predominant form of Spo11 contains exon 2 (Spo11-β) and the shorter
transcript is almost non-existent. Thus, it is possible that the two forms of Spo11 may
have different roles, e.g. catalytic and structural, that may be dependent on the presence
or absence of exon 2. We speculate that there is an additional role for Spo11, after it
generates DSBs, in stabilizing synapsis. Finally, we will present a progress report on the
biochemical characterization of Spo11 and on identifying additional protein cofactors that
interact with Spo11.
C2.20 - Coordinating chromosome pairing, synapsis and recombination during C.
elegans meiosis
A.M. Villeneuve, G.M. Chin, M. Colaiacovo, A.F. Dernburg, K.J. Hillers, A.J.
MacQueen, K. Reddy and G. Stanfield, Developmental Biology and Genetics, Stanford University
School of Medicine, Stanford, CA, U.S.A
We used genetic screens and a functional genomics strategy to identify components of
the machinery responsible for establishing and maintaining homolog associations during
meiotic prophase in C. elegans. One class of genes (chk-2, hal-2) is required both for
initial establishment of homolog pairing and for the major spatial reorganization of
chromosomes that normally accompanies the onset of pairing. chk-2 is also apparently
required for initiation of meiotic recombination. chk-2 encodes a C. elegans ortholog of
the Cds1/Chk-2 kinases previously implicated in replication block and DNA damage
checkpoints in other systems. We propose that chk-2 functions during premeiotic S phase
to enable chromosomes to become competent for subsequent meiotic prophase events
and/or to coordinate replication with entry into prophase. A second class of genes (sys-1,
13
sys-2 and sys-3) is required to stabilize and maintain homolog associations subsequent to
initial pairing. SYS-1, SYS-2 and SYS-3 are likely structural components of the
synaptonemal complex, consisting mainly of predicted coiled-coil domains. SYS-1 is
localized at the interface between synapsed chromosomes in pachytene nuclei. Crossing
over is severely reduced in sys mutants, indicating that initial homolog recognition and
pairing are insufficient to allow exchange and that synapsis is necessary to accomplish
crossover recombination. A pachytene checkpoint is triggered in these mutants, resulting
in frequent apoptosis of female germ cells.
We will also discuss experiments analyzing the meiotic behavior of fusion
chromosomes, which reveal both sequence-intrinsic features and chromosome-wide
mechanisms governing the distribution and frequency of crossovers.
C2.21 - Meiotic pairing and chromosome segregation in Drosophila oocytes
R. S. Hawley, S. Page and H. Matthies, Department of Genetics, Section of MCB, University of
California at Davis, Davis, California 95616
The talk will focus on three aspects of our studies of meiosis in Drosophila females. The
first of these will our studies of the C(3)G protein, a fly homologue of the yeast SC
protein ZIP1. Studies of the C(3)G protein in fixed ovarioles have provided new insights
into the timing and process of synapsis and into the organization of the chromosomes
within the nucleus. The second focus will be our recent work on the NOD protein, which
is required for the proper segregation of achiasmate chromosomes. Although the motor
domain of NOD has an ATPase activity typical of other kinesins, the binding affinity of
NOD to microtubules is not the tightest in the ATP state. Moreover, the sequence of
NOD is markedly different from that of other kinesins both in terms of critical residues in
the ATP and microtubule binding domain of kinesin and in terms of the absence of a neck
region. Directed amino acid substitutions in an otherwise functional kinesin protein,
which were designed to mimic the NOD sequence, result in full loss of motor activity but
retain a NOD-like ATPase activity. For these reasons we conclude that NOD is not a
motor protein in a conventional sense, but rather NOD functions as a brake that allows
the attachment of chromosome arms to microtubules in a fashion that counterbalances
the poleward forces being exerted at the centromere. Finally, we will present evidence
that the activity of NOD is regulated hormonally by a signal transduction pathway.
C2.22 - Telomere-led pairing precedes homologous synapsis in Arabidopsis thaliana
S J Armstrong, F.C.H. Franklin and G H Jones, School of Biosciences, The University of
Birmingham, Birmingham B15 2TT
An outstanding question is how homologous chromosomes pair, synapse and recombine
during meiosis. We are using FISH in combination with cytology and immunocyto-
14
chemistry to examine chromosome behaviour during early meiosis in wild type
Arabidopsis and in a meiotic mutant (asy1). Specific regions of chromosomes have been
marked with a combination of repeat and single copy sequences. This paper presents our
results for telomere behaviour during early meiosis. We have found that both in somatic
cells and in the S. phase preceding meiosis (marked by incorporation of BrdU and its
detection by BrdU antibodies) the telomeres have a characteristic organization. In both
cases the telomeres are clustered around the nucleolus. As the cell progresses into
leptotene this association disappears. The number of signals is decreased from around 20
to 10, indicating that the telomeres are undergoing pairwise association. At the same
time they disperse around the nuclear periphery, preceding any obvious pairing of the
homologues. When the chromosomes start to synapse the paired telomeres are
redistributed into a loose bouquet. This is disrupted at pachytene, as the telomeres again
distribute around the nuclear periphery. In the meiotic mutant, the gene ASY 1, which has
homology to yeast HOP1, is disrupted, and chromosome synapsis is not evident during
zygotene and pachytene. However, during premeiosis and leptotene, telomere behaviour
in asy1 is identical to wild-type. It is only later, during pachytene and diplotene that the
telomeres behave abnormally, reflecting the failure of homologous chromosome
synapsis.
C2.23 - Binding of premeiotic polycomb proteins by chromosome-specific sequences
in wheat – an implication for initiation of meiotic pairing
G. Grafi, B.Liu, F. Han, C. Melamed-Bessudo and M. Feldman, Dept. of Plant Sciences, The
Weizmann Institute of Science, Rehovot 76100, Israel
In accord with the currently growing view that initiation of meiotic pairing is mediated by
DNA-protein interaction, we obtained evidence pointing to the involvement of polycomb
proteins in this crucial event. About 20 different chromosome-specific sequences (CSSs)
were recently isolated, allocated to chromosomes, mapped and sequenced in polyploid
wheat. These low-copy, non-coding, highly conserved sequences are clustered in distinct
chromosomal regions. Because most DNA sequences are not homologue-specific in
polyploid wheat, the regions containing the CSSs are the main or even the only
homology-determining regions (HDRs). These regions which may correspond to the
classical pairing-initiation sites, are proposed here to mediate homology attraction and
initiation of meiotic pairing by acting as binding sites for pairing proteins. Here we
present data indicating that HDRs bind chromatin-associated, polycomb proteins during
early stages of meiosis. Sequence analysis showed that all CSSs possess high frequency
of polycomb-response element (PRE) with the core sequence GCCAT, which is the
polycomb protein binding site. PREs were shown to mediate the repressive function of
polycomb-group (PcG) proteins and induce “pairing-sensitive silencing” in Drosophila,
whereby alleles are brought close to one another via PcG-mediated interaction between
two homologous PREs. Band-shift assays showed the capacity of PREs found in CSSs to
specifically bind wheat premeiotic proteins; DNA-protein complexes could not be
detected at postmeiotic stage. Our results suggest that PREs clustered in HDRs may be
15
part of the molecular machinery that acts during meiosis to initiate pairing between
homologous chromosomes in bread wheat. Because PREs are characteristic of HDRs in
all chromosomes, their presence, although essential, is insufficient to ensure homologous
pairing. Presumably, other sequences within HDRs determine the specificity of
chromosome pairing. Isolation of PRE-binding proteins may lead to a better
understanding of the molecular mechanism underlying homologous recognition,
attraction and pairing initiation during meiosis.
C2.24 - The Ph1 locus reduces non-homologous centromere associations somatically
and meiotically
Enrique Martinez-Perez, Peter Shaw and Graham Moore, John Innes Centre, Norwich Research
Park, Colney, Norwich NR4 7UH, UK
Many plants, including wheat, are polyploid. The presence of more than one diploid set
of similar chromosomes can affect the correct assortment of homologous chromosomes
during meiosis. The Ph1 locus in hexaploid wheat restricts chromosome pairing and
recombination to true homologues. We show that centromeres associate in pairs prior to
meiosis in polyploid cereals, but not until the beginning of meiosis in their diploid
progenitors. These centromere associations have the potential to sort chromosomes into
homologous pairs before meiosis. Centromeres also associate in pairs during xylem
vessel cell development in the roots of hexaploid wheat but not in those of diploid
species. In these cells chromosomes become polytene. Centromere association is only
observed in certain developmental pathways of polyploid species. With centromeres
associating in different tissues we have designed an experiment to dissect the factors
controlling centromere associations. We have used a wheat/rye hybrid, where only nonhomologous chromosomes are present, to analyse the effect of Ph1 on centromere
associations. In the presence of Ph1 the level of non-homologous centromere association
is lower than in its absence. Further more, during meiosis the non-homologous
centromere association made premeiotically are corrected in the presence of Ph1 but not
in its absence. Therefore Ph1 is not responsible for the induction of centromere
associations but rather for its specificity.
C2.25 - The onset of meiosis in hexaploid wheat: The effect of the Ph1 locus on
chromatin organisation and homologous association
T. Naranjo and B. Maestra, Universidad Complutense, Madrid, Spain; J. H. de Jong, Wageningen
Agricultural University, The Netherlands; K. Shepherd, University of Adelaide, Australia
The organisation of centromeres and telomeres of wheat chromosomes and the behaviour
of two rye 5RL telocentrics added to wheat are analysed at premeiotic interphase and
16
early prophase I stages both in the presence and in the absence of the Ph1 locus using in
situ hybridisation. Prior to the bouquet formation centromeres concentrate at one pole of
the nucleus while telomeres spread on the opposite hemisphere. Centromere signals
numbers close to the haploid number (n = 22) confirm the occurrence of centromere
association at premeiotic interphase. However, in most cells of both genotypes, the rye
homologues occupy separated domains and their centromeres are not associated. The
bouquet formation starts at a stage reported earlier as premeiotic interphase. All
telomeres move to the bouquet site except those of the centromere end of the 5RL
telocentrics. Rye chromosomes decondense at this stage and appear confined to separate
territories in most cells of the wild-type line. In the ph1b mutant line, the rye
chromosome fibres are less folded, intermingled, and spread through the nucleus.
Subsequently, in the wild-type genotype, the rye chromosomes appear intimately
associated at the telomeric end and in some cases at both chromosome ends, which locate
at the bouquet suggesting that telomeres of the centromeric end were permitted to migrate
to the cluster site later than the noncentromeric telomeres. When the bouquet disorganises
most wild-type meiocytes show complete rye pairing. The ph1b mutant line, in addition
to a different chromatin organisation, shows a delay in the chromosome pairing
development.
C2.26 - Beyond the core transcriptome in budding yeasts
M. Bellis1, T. Orr-Weaver2, D. Fenger2, O. Ait-Ahmet3, R. Esposito4 and M. Primig3
1
CRBM, 1919, rte de Mende, 34293 Montpellier, France, 2Whitehead Institute, Nine Cambridge Center,
Cambridge, USA, 3IGH, 141, rue de la Cardonille, 34396 Montpellier, France, 4The University of Chicago,
920E 58th Street, Chicago, USA
Whole-genome expression profiling of spore development in budding yeast revealed the
strain-independent transcriptional induction of at least 900 “core” genes/ORFs during the
process. The data can be downloaded and/or accessed through a searchable relational
database (yMEx, yeast Meiotic Expression) that also comprehensively covers information
from global sources (e.g. SGD, MIPS) as well as other relevant expression studies. We
now have initiated genetic and in silico follow-up experiments to address two key
questions. Firstly, how many of the meiotically regulated genes/ORFs are essential for
spore development (in a strain independent manner)? Secondly, how many of these
genes are conserved between species and essential for gamete formation in higher
eucaryotes as well? Preliminary results obtained by an in silico screen of the D.
melanogaster genome using ~1500 yeast genes will be discussed.
Many promoters of the meiotically regulated core genes contain matches to
Abf1p, Ume6p/Ime1p and Ndt80p target sites. To better understand the biological
significance of these potential regulatory elements, we have initiated expression profiling
experiments comparing wild-type strains to ume6 deletion and abf1-1ts mutant strains
during mitotic growth.
17
C2.27 - Assembly and disassembly of rat SCs
C. Heyting, M. Eijpe and H.H. Offenberg, Wageningen University, The Netherlands; E.
Revenkova and R. Jessberger, Mount Sinai School of Medicine, New York
After premeiotic S-phase, the two sister chromatids of each chromosome develop a single
common axial structure, called axial element, which keeps the sister chromatids together
throughout meiotic prophase. The axial elements of homologous chromosomes are
incorporated as lateral elements into synaptonemal complexes. We have analyzed the
assembly and disassembly of axial elements in mouse and rat by immunocytochemical
labeling, using antibodies against the known axial element components SCP2 and SCP3,
and against mouse proteins that are homologous to components of the cohesin complex
(cohesins) of yeast. We found that in mouse and rat, proteins homologous to cohesins
Smc1 and Smc3 co localized with SCP2 and SCP3 in almost all stages of meiotic
prophase. Proteins homologous to other yeast cohesins however, occurred specifically in
axial elements long before SCP2, SCP3, SMC1 and SMC3, and persisted there longer.
The RAD50 protein, which is involved in the initiation of meiotic recombination,
appeared in high abundance in meiotic prophase cells simultaneously with or slightly
later than the first cohesins, but it did not co localize with them. RAD51, which is
involved in later steps of meiotic recombination, largely co localized with the axial
elements as defined by the earliest cohesins. We propose that recombination complexes
are initially formed throughout meiotic prophase chromatin, and are then transferred to
cohesins for subsequent steps in recombination.
C2.28 - Interactions of recombination proteins RAD51/DMC1, RPA and BLM in
mouse and rat synaptonemal complex associated recombination nodules
Peter B. Moens, Nadine Kolas, Madalena Tarsounas and Barbara Spyropoulos, York
University, Department of Biology, 4700 Keele str. Toronto ON, M3J 1P3
Recombination in the developmental pathway of meiotic prophase in yeast is initiated by
double strand breaks and exonuclease resection that results in single strand DNA tails
that are the substrate for RPA and RAD52 proteins. At those sites protein complexes
form which contain, in part, recombinases RAD51 and DMC1 and the numbers of foci
correspond to the numbers of recombinant events. From immunocytological observations
we report that in mouse and rat, unlike yeast, RPA expression follows rather than
precedes RAD51/DMC1 expression and that RPA invades and replaces RAD51/DMC1
foci. Subsequently, the chromosome-stability protein, BLM, becomes incorporated into
the foci. We rationalize that in organisms with large genomes such as lily and mammals
which have extensive duplications, homeologies, and repeated sequences, the complex
task of searching for proper homologies during early meiotic prophase requires large
numbers of RAD51/DMC1 homology-search and strand-invasion complexes, well in
18
excess of the number required for recombinant events, and that the excess of interacting
molecules are subsequently resolved without reciprocal recombination by the combined
action of RPA and BLM. We demonstrate that the electron microscope-defined late
recombination nodules which correspond to sites of reciprocal recombination, contain
RPA, presumably to facilitate the loading of additional proteins of the recombination
machinery such as MLH1.
C2.29 - Chromosomal pairing and segregation in mice lacking the synaptonemal
complex protein 3
C. Höög, J-G. Liu, J. Pelttari, M-R. Hoja and L. Yuan, Karolinska Instititutet, Stockholm,
Sweden
We have previously characterized a structural component of the axial element of the
synaptonemal complex (SC) encoded by a meiosis-specific protein called SCP3 (Yuan et
al., JCB (1998) 142:331-339). We have generated a null mutation in the SCP3 gene and
found that the homozygous mutant males become sterile due to a complete elimination of
spermatocytes (Yuan et al., (2000) Mol. Cell 5: 73-83). The mutant spermatocytes exhibit
extensive chromosomal asynapsis and undergo apoptosis. We have shown that the
apoptotic mechanism that responds to asynapsis in the absence of Scp3 is p53independent (Yuan et al., (2001) Cell Death and Diff., in press). We have now also in
detail analyzed the relative importance of the synaptonemal complex and the cohesin
complex for pairing of sister chromatids and homologous chromosomes during meiosis.
Cohesin complexes have been shown be important for sister chromatid pairing in mitotic
cells. We find that cohesin complexes associate to sister chromatids in early meiotic cells
in the absence of an axial element and that the axial elements have little influence on the
organization of cohesin complex structures and sister chromatid pairing. Our data also
suggests that cohesin complexes promote both recombination and homologous
chromosome pairing in meiotic cells in the absence of an axial element or a synaptonemal
complex. Finally, we find that SCP3 is required for the assembly of a second axial
element protein, SCP2, onto the meiotic chromosomes and that both proteins are required
for the formation of the axial element structure seen in vivo.
19
C2.30 - In vivo analysis of synaptonemal complex formation during yeast meiosis
E. White1, C. Cowan2, W.Z. Cande2, and D. B. Kaback3
1) Department of Microbiology and Molecular Genetics, UMDNJ-Graduate School of Biomedical
Sciences, Newark, NJ 07103, 2) Department of Molecular and Cell Biology, University of California,
Berkeley, Berkeley, CA 94720, 3) Department of Microbiology and Molecular Genetics, UMDNJ-New
Jersey Medical School, Newark, NJ 07103. (973) 972-4192
Meiotic pachytene is marked by the appearance of the synaptonemal complex (SC), a
tripartite structure believed to play a role in regulating pairing and recombination
between homologous chromosome pairs. The ZIP1 gene encodes an essential protein of
the central region of the SC. To study the dynamics of SC formation in living cells, a
ZIP1-GFP fusion was constructed and used to replace the wild type ZIP1 gene in
diploids. The fusion complemented a zip1 deletion to ~80% of wild type activity and
produced normal appearing SC as judged by electron microscopy. Kinetics of SC
formation and dissociation were in agreement with previous studies. Deconvolution light
microscopy revealed that the SC was assembled at the nuclear periphery and then
assumed a more random distribution throughout the nucleus. The effects of several
meiotic mutants on SC formation will be discussed.
C2.31 - Sgs1 regulates meiotic homolog pairing in yeast
Beth Rockmill, Steve Branda and Shirleen Roeder, Yale University HHMI, New Haven CT,
USA
The sgs1 mutant undergoes a partial checkpoint arrest in meiosis. Those cells that
complete meiosis have enjoyed full levels of recombination, but are somewhat inviable
(65%) due to chromosome missegregation. Both the arrest phenotype and spore
inviability are not dependent on recombination. The arrest is suppressed by certain DNA
damage checkpoint mutations (ddc1, rad24 and mec3), and these mutations partially
suppress the segregation defect as well.
Chromosomes in the zip1 mutant cannot synapse, but they form axial cores that
are paired and connected at several sites called axial associations (AAs). AAs are sites of
accumulation of proteins involved in synapsis initiation and crossing over. The sgs1 zip1
double mutant forms an excess of AAs causing chromosomes to appear synapsed. The
Sgs1 protein localizes to foci along synapsed chromosomes, corresponding to AAs.
Homolog pairing occurs in the absence of recombination. For example, mer2 strains
undergo reduced, but significant levels of meiotic homolog pairing (assayed by FISH).
An ndj1 mer2 double mutant is further reduced for homolog pairing. ndj1 mutants do not
form chromosome bouquets, which are thought to participate in chromosome alignment.
sgs1 mer2 diploids display a higher level of pairing than mer2 strains, and this improved
pairing is partially dependent on Ndj1. An ndj1 mutation also partially suppresses the
sgs1 segregation defect, suggesting the involvement of paired chromosomes in
chromosome missegregation.
20
Our model for the role of Sgs1 in processing paired chromosome intermediates
will be presented.
C2.32 - The roles of proteolysis in separating sister chromatids during mitosis and
meiosis
Kim Nasmyth, Frank Uhlmann, Sarah Buonomo, Attila Toth and Kirsten Rabitsch, IMP,
Dr. Bohr-Gasse 7, A-1030 Vienna, Austria
In eukaryotic cells, replicated DNA strands remain physically connected until their
segregation to opposite poles of the cell during anaphase. This "sister chromatid
cohesion" is essential for the alignment of chromosomes on the mitotic spindle during
metaphase. Cohesion depends on a multisubunit protein complex called cohesin, which
possibly forms the physical bridges that connect sisters. Proteolytic cleavage of cohesin's
Scc1 subunit at the metaphase to anaphase transition is essential for sister chromatid
separation and depends on a conserved protein called separin, which is a cysteine
protease related to caspases. Cleavage of Scc1 in metaphase arrested cells is sufficient to
trigger the separation of sister chromatids and their segregation to opposite cell poles.
It has been proposed but never proven that cohesion between sister chromatids
distal to chiasmata is responsible for holding homologous chromosomes together while
spindles attempt to pull them towards opposite poles during metaphase of meiosis I.
Meanwhile, the mechanism by which disjunction of homologues is triggered at the onset
of anaphase I has remained a complete mystery. In yeast, cohesion between sister
chromatid arms during meiosis depends on a meiosis-specific cohesin subunit called
Rec8 which replaces Scc1. Cleavage of Rec8 by separin at one of two different sites is
necessary for the resolution of chiasmata and the disjunction of homologous
chromosomes during meiosis.
The orderly reduction in chromosome number that occurs during meiosis depends
on two crucial aspects of chromosome behaviour that are specific to the first meiotic
division. These are the retention of cohesion between sister centromeres and their
attachment to microtubules that extend to the same pole (monopolar attachment). We
have identified a centromere associated protein called Mam1 which is essential for
preventing bipolar attachment during meiosis I. We also show that the meiosis-specific
cohesin, Rec8, is essential for maintaining cohesion between sister centromeres but not
for monopolar attachment. We conclude that monopolar attachment of sister kinetochores
during meiosis I requires a meiosis-specific protein and is independent of the process that
protects sister centromere cohesion. Future work must address the mechanism by which
Mam1 co-orients sister kinetochores and the mechanism that protects Rec8 in the vicinity
of centromeres from separin at the first meiotic division.
21
C2.33 – Sister chromatid cohesion during meiotic divisions in mouse spermatocytes
J.A. Suja1, N. Pezzi2, I. Prieto2, M.T. Parra1, L. Kremer2, J.S. Rufas1 and J.L. Barbero2
1
Dept. Biología, UAM, Madrid, Spain. 2Dept. Immunology and Oncology, CNB, Madrid, Spain
We have analysed by immunofluorescence the spatio-temporal expression of STAG3 in
mouse spermatocytes. STAG3, a meiosis-specific subunit of the cohesin complex of
mammals, is first detected in prezygotene cells. Then it localises along axial elements
(AEs) during pairing and synaptonemal complexes during pachytene. In diplotene cells
there is a partial loss of STAG3 at desynapsed lateral elements but it still persists at the
interchromatid domain in the arms and weakly at homologous centromeres in metaphase
I bivalents. This labelling disappears throughout anaphase I when sister arm cohesion is
lost, and is no longer revealed during meiosis II. These results suggest that STAG3 is a
component of AEs that mainly ensures sister-chromatid arm cohesion during mammalian
meiosis I. Moreover, our results indicate that the two-step loss of STAG3 may be
independent of the APC-separin pathway. Since the expression of STAG3 during meiosis
I is similar to that previously reported for SCP3, a protein appearing at AEs of rodents
with a suggested role in maintaining sister cohesion, we colocalised both proteins.
Results show that both proteins colocalise at the interchromatid domain of metaphase I
bivalents, and are released from arms during the metaphase/anaphase I transition.
However, their distribution and release from centromeres is different. SCP3 shows a
complex unpredicted 3D distribution, and unlike what it was previously reported, it
dissociates from the centromere during telophase I. We propose that SCP3 may ensure
sister-kinetochore cohesion that permits an accurate biorientation of the bivalent.
C2.34 - Functional analysis of Arabidopsis cohesins
A.M. Bhatt, J. Costa-Nunes, Y. Li and H.G. Dickinson, University of Oxford, Oxford, U.K
The Arabidopsis genome encodes most components of the cohesin complex. Unlike
yeast, Arabidopsis has four different REC8/RAD21 homologs [1,2] - DIF1/SYN1,
AtRAD21-1, AtRAD21-2 and AtRAD21-3, all with conserved N and C terminal domains,
but of different sizes (617, 809, 693 and 1031 amino acids respectively). The functional
analysis of these, DIF1 (determinate, infertile 1), with a function essential for meiosis,
and AtRAD21-1, AtRAD21-2, AtRAD21-3, with functions that are currently unknown,
will be presented. Although the DIF1 gene is expressed during mitosis, dif1 mutants have
no obvious vegetative phenotype. In dif1 mutants the defects in meiosis, are first evident
by metaphase I, when dif1 pollen mother cells show extensive chromosome
fragmentation and univalents. DIF1 specific antibodies and GFP fusions are being used to
analyse the localisation of DIF1. Reducing the expression of AtRAD21-1 through antisense expression has no effect on transgenic plants, suggesting that AtRAD21-1 is not
essential for mitosis or meiosis. Other putative components of the Arabidopsis cohesin
complex have also been identified and cloned; these include Arabidopsis homologs of the
SMC1 and SMC3 gene. The meiotic functions and interactions of these putative cohesin
genes is being analysed through the isolation of transposon insertions in each of them.
22
REFERENCES - [1] Parisi et al., (1999) Mol. Cell. Biol. 19, 3515-3528. [2] Birkenbihl,
R.P. and Subramani, S. Nucleic Acids Res. 20, 6605-6611.
C2.35 - Regulation and function of genes controlling chromosome segregation in
yeast
R.E. Esposito, B. K. Washburn, M. Primig, R. Williams and G.G. Tevzadze, University of
Chicago, Chicago, IL 60637 USA
Meiosis and spore formation in budding yeast is regulated by a transcriptional program
resulting in the coordinated expression of at least seven classes of genes detected by
recent DNA array analysis. A critical regulatory switch controlling early gene expression
requires Ume6. This DNA-binding protein represses early genes in mitosis through
interaction with the Sin3/Rpd3 histone deacetylase repressor complex, and activates the
same genes in meiosis by association with Ime1. Mutational analysis of Ume6 reveals
that conversion of the complex from a repressor to an activator involves a two step
process in which Ime1 i) relieves Sin3/Rpd repression and ii) provides an activation
domain to Ume6. Based on these studies we have constructed an Ime1 independent
artificial activator for meiosis using the Gal4 activation domain fused to Ume6 mutations
which disrupt Sin3/Rpd3 binding. DNA array analysis of Ume6 mutants further reveals
that it also participates in regulation of some middle and late genes. Two early genes
under Ume6 control extensively studied in our lab encode Spo1 (a phospholipase B
homolog required for SPB duplication and subsequent events) and Spo13 (a nuclear
phosphoprotein required for centromere cohesion and proper segregation at MI). Several
other genes controlling chromosome segregation and formation of spores have also been
identified that are expressed in mid and late meiosis which suggest the existence of a
novel signaling pathway coupling progression of the divisions and packaging of meiotic
products into spores. The functions and dependency relations of these genes during
sporulation will be discussed.
C2.36 - The spindle checkpoint in meiosis
Marion Shonn*§, Robert McCarroll# and Andrew Murray*
* Department of Molecular and Cellular Biology, Harvard University, Cambridge MA, § Department of
Biochemistry, Univeristy of California, San Francisco, # Celeron Genomic, Cambridge, MA.
The spindle checkpoint prevents cells with mis-aligned chromosomes from starting
anaphase. We have shown that the budding yeast spindle checkpoint plays a critical role
in meiotic chromosome segregation. Spindle checkpoint mutants (mad2∆) undergo high
levels of meiosis I non-disjunction and the frequency of these events increases with
23
increasing chromosome length. Non-disjunction in mad2∆ can be prevented by delaying
progression of the metaphase to anaphase transition.
We have investigated a second role of the spindle checkpoint in meiosis I, namely
in promoting bipolar attachment. The kinetochores of linked homologs attach to opposite
poles of the spindle during metaphase thereby ensuring that homologs will segregate
away from one another in anaphase. We assayed bipolar attachment by marking the
centromeres of homologous chromosomes with GFP and determining the position of the
centromeres with respect to the metaphase I spindle. If homologous centromeres make a
bipolar attachment, they are pulled towards opposite poles and the GFP signals are
separated. In wild-type cells, the majority of metaphase I cells showed separated
homologous centromeres, indicating that the homologs had achieved bipolar attachment.
In contrast, mad∆ homozygous mutants showed a significant decrease in the number of
metaphase I cells with separated homologous centromeres. The observations suggest that
the spindle checkpoint promotes bipolar attachment in metaphase I.
C2.37 - The origin of human nondisjunction
Terry Hassold, Case Western Reserve University, 10900 Euclid Avenue, Cleveland OH44106, USA
Trisomy is the most commonly identified chromosome abnormality in humans, occurring
in at least 4% of all clinically recognized pregnancies; it is the leading known cause of
pregnancy loss and of mental retardation. Over the past few years genetic mapping
studies have led to the identification of the first molecular correlate of human
nondisjunction; i.e., altered levels and postioning of meiotic recombinational events. In
initial studies of trisomies 16 and 21, we observed an increase in 0 exchange events or in
distal-only or pericentromeric exchanges in nondisjunction-generating meioses. As these
events occurred in similar frequencies in nondisjunctional meioses of younger and older
women, we suggested that maternal meiotic nondisjunction required "two hits": first, the
establishment of a "vulnerable" bivalent in prophase I (in the fetal ovary) and second,
abnormal processing of the bivalent at metaphase I (following ovulation in the adult); the
second event was suggested to be the age-dependent factor. We will update the data that
led to this model, which remain consisent with our original interpretations. However, we
will also present data from other human trisomies that indicate chromosome-specific
nondisjunctional mechanisms; i.e., patterns differing from those associated with trisomies
16 and 21, suggesting that the mechanisms are more complicated than we originally
assumed.
In addition to studies of the mechanism of origin of trisomy, we will discuss
preliminary epidemiological studies of trisomy 21, designed to identify significant
intrinsic or extrinsic etiological agents. Additionally, we will sumarize initial murine
studies intended to model certain aspects of human nondisjunction, analyzing meiosis in
female carriers of paracentric inversions and early mitotic divisions in embryos carrying a
nondisjunction-prone Y chromosome.
24
C2.38 - Meiotic recombination and segregation in human translocation carriers
M A Hulten, Department of Biological Sciences, University of Warwick, Coventry CV4 7Al,
[email protected], A.S.H. Goldman, Department of Molecular Biology and Biotechnology,
University of Sheffield, S J Armstrong, School of Biosciences, The University of Birmingham
Human translocation carriers are most often ascertained because of impaired reproductive
performance including infertility and subfertility (miscarriages, intrauterine or perinatal
deaths and multiply malformed/mentally handicapped children). In human males, the
incidence of translocation heterozygosity is inversely proportional to sperm count; thus,
approximately 6 % of men with severe oligozoospermia are carriers. A vital question
with respect to fertility treatment is the risk of producing chromosomally unbalanced
gametes. This risk is dependent on the behaviour of the translocation during meiosis. In
this overview we describe segregation analysis and calculation of gametic output; these
were made possible, for the first time, by application of fluorescence in situ hybridisation
(FISH). These investigations confirm that translocation quadrivalents are abundant at MI
(no unequal bivalents found in any carrier) and that there is a general tendency for
increased chiasma formation within the interstitial segments. Interstitial segments form
chiasmata even when very short and under normal circumstances would have had few, if
any, chiasmata. A high proportion of MII spermatocytes contains unequal dyads. From
our data we have calculated MI segregation patterns and gametic output for individual
carriers. By far the largest mode of segregation is alternative/adjacent I (indistinguishable
after chiasma formation within the interstitial segments) in the order of 70 %, while
adjacent II and 3:1 segregations are infrequent. The proportion of genetically unbalanced
spermatozoa is high, around 60-70%, in comparison to those that are normal (15-20%) or
balanced (15-20%). FISH studies on sperm nuclei substantiate these estimates, indicating
a significant risk of producing unbalanced embryos by the currently most common
fertility treatment, intracytoplasmic sperm injection (ICSI).
C2.39 - The molecular regulation of cell identity and division in the male germline of
Arabidopsis
H.G. Dickinson and C. Canales, University of Oxford; R. Scott, University of Bath
Little is known of the genes regulating sex-cell specification in the anthers (male) and
ovules (female) of flowering plants. In contrast to other cell lines which arise from
“conventional” meristems (i.e.the shoot apical meristem), the male germline is generated
from four columns of archesporial cells which undergo a series of divisions giving rise to
three specialised wall layers (including the tapetal or nurse cells), and the sporogenous
(germline) cells themselves. The MADS-box gene NOZZLE (NZL) [1] has been shown
to play an important part in early archesporial function in the model plant Arabidopsis
thaliana, but mutant studies point to cell number and identity in both the wall and
25
sporogenous layers being regulated – at least in part – by the EXTRA SPOROGENOUS
CELLS (ESP) sequence.
Following meiosis I and II, the male meiotic products are segregated via a unique
type of division (tetrad) involving genes specific to meiosis, including TETRASPORE
(TES), which is involved in the synthesis of the callose cross-walls of the tetrad [1,2].
The nature of the ESP1 and TES gene products, together with their proposed
functions, will be discussed in the context of current models of meiotic initiation and
progression in multicellular eukaryotes as well as, more generally, for the specification of
plant cell lines.
[1].
Schiefthaler U. et al. Proc. Natl. Acad. Sci. USA 96: 11664-11669.
[2]
Spielman, M. 1997 Development 124, 2645-2657
[3]
Scott et al. 1998 Development 125, 3329-3341
C2.40 - Female mammalian meiosis I possesses a spindle checkpoint
H.A. Homer, A. Mc Dougall, A.P. Murdoch and M. Herbert, Reproductive Medicine,
BioScience Centre, International Centre for Life, Times Square, Newcastle upon Tyne, NE1 4EP
In mitosis, the spindle checkpoint ensures accurate chromosome segregation. Although it
is generally held that spindle checkpoint function in female meiosis I is deficient, the
evidence is conflicting. Moreover, there are no reports directly investigating the effects of
spindle damage on both anaphase I and MPF activity in oocytes.
In vertebrate mitotic cells, chromatid arms separate by a non-cleavage mechanism
when the checkpoint is active during a so-called C-mitosis. As abrogation of arm
cohesion is responsible for homologue segregation in meiosis I, might homologues
disjoin with time in the absence of a spindle (undergo a “C-meiosis”).
We show that spindle depolymerisation in female murine meiosis I prevents both
anaphase I and MPF inactivation. Furthermore, homologues do not undergo a “Cmeiosis” suggesting that the non-cleavage prophase pathway by which arm cohesion is
lost in vertebrate mitosis is not the primary mechanism for loss of arm cohesion in
meiosis I. We propose that this reflects checkpoint activity induced by free kinetochores,
and that homologue disjunction might be coupled to MPF inactivation. We suggest that
the long M phase of mammalian oocytes may be important to provide sufficient time for
chromosomes to align.
C2.41 - Meiotic sex chromosome inactivation and the sex body in mammalian
meiosis
P. S. Burgoyne, S. K. Mahadevaiah and J. M. A. Turner, National Institute for Medical
Research, London, UK
26
During the pachytene stage in male mammals, the XY bivalent lies within a discrete
structure termed the XY- or sex body. In the past, this structure has been viewed simply
as the morphological manifestation of the chromatin changes associated with meiotic sex
chromosome inactivation (MSCI), a process that has been suggested to be
mechanistically related to somatic X inactivation and to be mediated by Xist transcripts
(1). The function(s) of MSCI and sex body formation have been thought to relate to the
protection of the non-PAR X and Y axes from recombination and/or checkpoint
recognition. The present paper will describe studies of MSCI and/or sex body formation
in mice with a variety of sex chromosomally aberrant genotypes including: (a) XY
females in which MSCI and sex body formation are absent, (b) XY males with targeted
Xist mutations in which MSCI and sex body formation occur, and (c) XYY males in
which MSCI and sex body formation is disturbed. It will be argued that MSCI is initiated
prior to meiotic prophase, is independent of Xist, and may be an unfortunate consequence
of chromatin changes needed to focus the ‘homology search’ on the homologous
pseudoautosomal regions (PARs) (2). Sex body formation occurs later, coincident with
the recruitment of further proteins to the chromatin associated with the non-PAR X and Y
axes, and one or more of these proteins may play a role in masking this chromatin from
checkpoint recognition (2).
1. Ayoub et al. (1997). Chromosoma 106, 1-10.
2. Turner et al. (2000). Chromosoma 109, 426-432.
C2.42 - Colouring in meiosis of hybrids and mutants
G. Jenkins, University of Wales, Aberystwyth, UK
Fluorescence in situ hybridisation (FISH) with dispersed and tandemly repeated DNA
sequences has been used to probe meiosis in two higher plants challenged by hybridity
and mutation. A moderately repetitive, dispersed repeat has been isolated from Lolium
perenne (Poaceae), which is considerably more abundant in the genome of this species
compared with its close relative L. temulentum. Probing homoeologous chromosomes
during meiosis I of the F1 hybrid between these two species shows that this sequence
appears not to interfere with the structural integrity or recombination potential of hybrid
bivalents. The sequence discriminates between chromosomes of different parental origin
and enables tracking of the intimate interactions between homoeologues throughout
meiotic prophase. Hybrid bivalents are potentially useful substrates for comparative
bivalent mapping of both high and low copy DNA sequences, and may provide insights
into the construction of synaptonemal complexes and the consequences of genome
expansion.
The nuclear dispositions of two chromosomal domains were tracked during premeiosis and meiosis I in wild-type and two asynaptic mutants of rye (Secale cereale), by
means of simultaneous FISH with a subtelomeric tandem repeat and a pericentromeric
sequence. At the onset of meiosis in wild-type and asynaptic mutant sy9 these domains
form a typical bouquet conformation which persists until later in leptotene and zygotene
when it disperses. In contrast, asynaptic mutant sy1 fails to form comparable clusters in
27
half of its nuclei, and pericentromeres disperse prematurely in the majority of nuclei.
This mutant phenotype could arise from disturbances in the nuclear organisation of these
chromosomal regions.
C2.43 - Chromosome versus genic mechanisms of hybrid sterility
P.M. Borodin, M.B. Rogatcheva, A.V. Polyakov, Institute of Cytology and Genetics,
Novosibirsk, Russia; S.I. Oda, Nagoya University, Japan; C.R. Bonvicino, National Institute of
Cancer, Rio de Janeiro, Brazil; P.S.D. Andea, Institute Oswaldo Cruz, Rio de Janeiro, Brazil
Sterility of hybrids between karyotypically diverged species or chromosomal races within
a species is usually interpreted as evidence of the chromosomal mechanism of
reproductive isolation. However, the data accumulated on mammalian hybrids show that
chromosomal divergence played a minor if any role in hybrid sterility. Although sterile
hybrids often suffer meiotic pairing problems we found no correlation between a degree
of chromosomal divergence and meiotic disruption in geni Mustela and Calamiscus:
some species producing sterile hybrids did not differ in chromosomal arrangement, while
some karyotypically diverged species of the same genus produced fertile hybrids.
Analysis of the hybrids between chromosome races of Sorex araneus carrying a complex
meiotic configuration (chain-of-IX) demonstrated an orderly chromosome pairing and
nearly normal fertility. We analysed a role of chromosomal and genic mechanisms of
hybrid sterility in hybrids between two chromosome races of Suncus murinus which
differed for 7 chromosome rearrangements. We found that meiosis in sterile hybrids did
not progress beyond pachytene and was severely disrupted. However, there was no
correlation between the number of heterozygous rearrangements per individual and its
fertility. Therefore we concluded that meiotic arrest and sterility of interracial hybrids
was not determined by structural heterozygosity. The male sterility in this case was
shown to be controlled by a single gene. Thus, we suggest that meiotic problems in
sterile hybrids are usually determined by incompatibility of genetic systems controlling
chromosome pairing and segregation, rather than chromosomal incompatibility.
C 2.44 - GCN4 de-repression and genetic recombination in S.cerevisiae
MFF Abdullah and RH Borts, Dept. Genetics, University of Leicester, Leicester LE1 7RH, UK
In S.cerevisiae, the transcription factor Gcn4p stimulates the expression of amino acid
biosynthetic enzymes in at least 12 different pathways. GCN4 itself is regulated by
amino acid availability, being repressed under non-starvation conditions and de-repressed
when one or more amino acid is lacking. While studying genetic recombination of the
his4:xho allele , we noticed a 1.5 - 2.5 fold increase in gene conversion in strains where
the combination of auxotrophic markers and nutrient supplement is likely to lead to
28
GCN4 de-repression. This increase was not observed when we repeated the experiments
in Dgcn4 mutants of the same strains. Furthermore, in "repressed" strains engineered to
express Gcn4p constitutively, gene conversion levels increased 4-fold. We also observed
an increase in crossing-over in the HIS4-LEU2 interval in wildtype strains when
compared to Dgcn4 mutants. The HIS4 gene has five Gcn4p binding sites in the
promoter region, where double-strand breaks are thought to be initiated. We suggest that
derepression of GCN4 expression can lead to increase gene recombination activity at the
HIS4 locus, by increased binding of GCN4p to these sites.
C2.45 - Mutagenesis of ScMLH1: Implications for ATPase domain function and
Pms1p and Mlh3p interaction domains for meiotic recombination
E.R. Hoffmann and R.H. Borts, Dept. of Genetics, University of Leicester, University Road,
Leicester, LE17RH
The mismatch repair protein Mlh1p, is involved in post-replicative mitotic repair as well
as gene conversion during meiosis. In addition, Mlh1p also promotes crossing over
during meiosis and mutants deficient in Mlh1p show decreased meiotic viability with an
overall increase in two- and zero- spore tetrads, indicative of meiosis I non-disjunction.
The protein shows high homology to the E.coli MutL protein in the N-terminus- the
putative ATPase domain. The C-terminus shows little homology to MutL as well as the
other three yeast MutL homologs Pms1p, Mlh3p, and Mlh2p.
We have carried out point mutagenesis on Mlh1p, knocking out amino acid
residues in the highly conserved GFRGEAL terminus located in the N-terminus as well
as in the conserved C-terminal homology domain. We find that within the GFRGEAL
box, amino acid changes affecting mismatch repair similarly, may affect crossing-over
differentially. Some of the Mlh1p point mutants also indicate that both crossing-over
decreases as well as loss of mismatch correction ability influences meiotic viability.
Finally, Mlh1p mutants unable to interact with Pms1p, and hence not form heterodimers,
also show a meiotic crossing over deficiency in addition to a decrease in gene conversion.
As pms1 deletion mutants have no meiotic crossing over phenotype, Mlh1p may use
similar interactions with Mlh3p to form heterodimers during meiotic crossing over.
C2.46 - DNA mismatch repair and meiotic homeologous recombination
Lynne A. Rainbow, Scott R. Chambers and Rhona H. Borts, Department of Genetics,
University of Leicester, LE1 7RH
One effect of the mismatch repair system is to limit exchanges between divergent DNA
sequences. This effect has been examined in a partial hybrid of S. cerevisiae and the
closely related species S. paradoxus, wherein is found one copy of chromosome III from
S. paradoxus in an otherwise S. cerevisiae background. In the wild type partial hybrid,
29
tetrad analysis shows meiotic recombination is reduced 25 fold in four genetic intervals
along chromosome III, compared with the wild type homozygote. Certain mismatch
repair mutant partial hybrids (pms1,msh2 and msh6) partially restore the levels of meiotic
recombination (but not to the levels seen in the wild type homozygote). Other genes
appear to promote exchange between the homeologs (msh3, and possibly mlh2 and
hsm3). Epistasis analysis has revealed that these appear to act upstream of msh6.
There appears to be more than one mechanism involved in crossing over, and the
genetic requirements of these are different, for example, msh6 is not involved in the half
crossover pathway. Certainly the role of the mismatch repair system during homeologous
recombination is very complex.
During the course of these studies it was found also that Msh2 and Msh3 proteins
play a role in sister chromatid exchange which is not dependant on the presence of the
homologous chromosome. The role of the other mismatch repair proteins in this process
is being explored.
C2.47 - Recombinational DNA double strand breaks in mice precede synapsis
S. K. Mahadevaiah, J. M. A. Turner and P. S. Burgoyne, National Institute for Medical
Research, London, UK; F. Baudat, M. Jasin and S. Keeney, Memorial Sloan-Kettering Cancer
Center, New York, USA; E. P. Rogakou and W. Bonner, National Institutes of Health, Bethesda,
USA; P. de Boer, Institute of Animal Sciences, Wageningen, The Netherlands; J. Blanco-Rodríguez,
School of Medicine, Valladolid University, Spain
In Saccharomyces cerevisiae, meiotic recombination is initiated by Spo11-dependent
double-strand breaks (DSBs), a process that precedes homologous synapsis (1), but there
is no consensus as to the relative timing of these two events in mouse meiosis. In S.
cerevisiae, Drosophila melanogaster and mammalian cells, radiation-induced DSB
formation results in the rapid phosphorylation of histone H2AX, yielding a specific
modified form, γ-H2AX, that forms en masse at the sites of DSBs (2). We show that, in
non-irradiated male mice, γ-H2AX is present specifically in the testis. During leptotene
and zygotene, γ-H2AX antibody labels multiple chromatin domains that encompass those
stretches of axial elements that have foci of the recombinase DMC1. Using a recently
reported Spo11 mutant (3) we show that, these leptotene/zygotene γ-H2AX signals are
Spo11-dependent and disappear as synapsis progresses. Synaptic failure involving the
autosomes is correlated with a failure to resolve DSBs, as judged by the persistence of γH2AX signal on the chromatin around asynapsed axial elements; if asynapsed regions
subsequently achieve non-homologous synapsis, the γ-H2AX signal is lost. The
chromatin associated with asynapsed regions of the sex chromosomes is also γ-H2AX
positive and remains so until the end of diplotene; however, this positivity is largely
Spo11-independent. These data provide compelling evidence that, recombination in the
mouse is initiated by Spo11-dependent DSBs that form during leptotene; the processing
of these breaks is closely co-ordinated with synapsis.
1. Roeder, G.S. (1997). Genes Dev. 11, 2600-2621.
2. Rogakou, E.P. et al. (1999). J. Cell Biol. 146, 905-915.
30
3. Baudat, F. et al. (2000). Mol. Cell 6, 989-998.
C2.48 - Post-DSB recombination and SC formation occur by linked parallel
pathways
V. Börner and N. Kleckner, MCB, Harvard University, Cambridge, USA
Meiotic recombination, synaptonemal complex (SC) formation and cell cycle progression
are intimately coordinated in many organisms. In budding yeast, all three processes are
dependent on meiosis-specific genes (DMC1, MER3, ZIP2, MSH5, ZIP3, ZIP1) whose
primary defects emerge after formation of DSBs and axial elements. Systematic analysis
of these “intermediate block functions” (IBFs) in two strain backgrounds (SK1, BR) at
two temperatures reveals a single common picture:
SC formation is defective at both temperatures, though significant SC levels are
seen in all mutants except zip1. Recombinational progression is defective at high
temperature and distinctive mutant blocks define an ordered sequential pathway. At low
temperature, recombination proceeds without distinctive blocks and crossovers occur at
50% the wild type. Cell cycle arrest is triggered at high temperature only.
Conclusions: (1) IBFs are required for normal SC formation, presumably via
modulation of late leptotene axis status. (2) Linkage between DNA recombination and
axial changes is normally established via IBFs working collaboratively, irrespective of
ensuing progression. In the absence of one or more IBFs, temperature suffices to create
the linked situation but cannot substitute for IBFs in promoting recombination events
Recombination blocks reflect direct roles of the corresponding proteins. (3) At low
temperature, recombination in IBF mutants is unlinked from axial changes (“free
running”). (4) We propose that, in the linked situation, axial changes govern (a)
progression of recombination from stage to stage and (b) crossover control. (5) Cell cycle
arrest results from recombination defects, not defective SC formation.
C2.49 - Regions of homology introduced into homoeologous chromosomes, effect the
distributive segregation system in yeast
G. Gobert, P. Prestwich and A.S.H. Goldman, Molecular Biology and Biotechnology, University
of Sheffield, England
The distributive segregation of a Saccharomyces cerevisiae chromosome V from a
Saccharomyces carlsbergensis chromosome V has been assessed in two different
contexts. In the first, segregation was assessed by tetrad dissection when the S.
cerevisiae chromosome V was marked URA3 can1-R, and the S. carlsbergensis
chromosome was marked ura3 CAN1-S. Colonies from two spore viable tetrads that can
grow on uracil dropout plates but are sensitive to canavonine indicate a first meiotic non–
disjunction event. In the second context the S. cerevisiae chromosome V was inserted
31
with an allele of arg4. A second but different arg4 allele was inserted in either
chromosome III or VIII. Ectopic recombination between the two alleles can yield ARG+
spore colonies either through gene conversion alone, or with reciprocal exchange which
creates translocation chromosomes. Reciprocal recombination between a chromosome
VIII and the S. cerevisiae chromosome V has the potential to form a first metaphase
multivalent. In this case segregation of the chromosomes was followed by pulsed field
gel electrophoresis and southern analysis of ARG+ colonies derived from random spores.
The data suggest that an ectopic reciprocal recombination event disrupts the distributive
segregation system, even though we find such crossovers may not direct segregation
themselves.
C2.50 - The importance of nuclear localisation on the ability of allelic sequences to
recombine during meiosis
H. Schlecht and A.S.H. Goldman, Molecular Biology and Biotechnology, University of Sheffield,
England
The efficiency of ectopic recombination between inserts on heterologous chromosomes is
related to telomere proximity (Goldman and Lichten unpubl. obs.). The relative
improvement in the ability of sequences on heterologues to find each other when close to
a telomere could be related to the localisation of telomeres at the nuclear envelope, and is
possibly enhanced by the bouquet arrangement. We are testing this relationship two
ways. Firstly we are looking at the effect of bringing interstitial sequences to the nuclear
envelope, both by creating reciprocal translocations and using a molecular hooking
mechanism. Secondly we are testing the effect of mutants, which have a role in telomere
tethering to the nuclear envelope. Deletion of NDJ1, a meiosis specific telomere
associated protein, results in failure to form the bouquet arrangement and increases
ectopic recombination efficiency 2-fold. HDF1 is involved in telomere tethering to the
nuclear periphery prior to meiosis. Monitoring ectopic recombination efficiency in hdf1
and hdf1/ndj1 double mutants will allow us to assess the relevance of pre–meiotic/early
meiotic telomere localisation interactions between heterologous chromosomes. This in
turn will help indicate the influence normal telomere localisation has on chromosome
pairing.
32
C2.51 - STAG3 is involved in meiotic cohesin complex
N. Pezzi1, I. Prieto1, J.A. Suja2, L. Kremer1, C. Martínez-A1. J.S. Rufas 2 and J.L.
Barbero1
1
Dept.Immunology and Oncology, CNB, Madrid , Spain. 2 Dept. Biología, UAM, Madrid, Spain
We have previously isolated and characterized a new family of nuclear proteins called
stromal antigens or stromalins (SA/STAG) that are present from yeast to human. In
mammals, there are three members STAG1, STAG2 and STAG3 that show high
sequence identity degree. Recently, it has been found that STAG1 and STAG2 are
subunits of cohesin complexes in somatic cells. We reported that STAG3 is specifically
expressed in germinal cells and is located at the synaptonemal complex in prophase I,
but its function in meiosis remains unknown. The present study was focused on the
specific contribution of STAG3 in the chromosome pairing and segregation in
mammalian meiosis. Immunofluorescence analysis shows that STAG3 is present in
prophase I spermatocytes from leptotene stage and colocalizes with SCP3 in all bivalents
including the XY pair suggesting that STAG3 is located at the axial/lateral elements of
synaptonemal complex. STAG3 signal in metaphase I cells stains specifically the
interchromatid domain and is absent in the chiasma region. STAG3 disappears in the
metaphase/anaphase I transition and is not detected in later stages of meiosis.
Immunoprecipitation experiments with anti-STAG3 antibodies shows its interaction with
the structural maintenance of chromosome proteins SMC1 and SMC3. Based on these
results, we propose that STAG3 is a sister chromatid arm cohesin specific of mammalian
meiosis I as component of a meiotic cohesin complex associated with SMC1, SMC3 and
REC8 proteins.
C2.52 - Sister chromatid cohesion in meiosis
Beth M. Weiner, Dogan Perese, Job Dekker and Nancy Kleckner, Harvard University,
Cambridge, MA USA
PDS5 is an essential gene in Saccharomyces cerevisiae. The protein has been shown to
be important for chromosome structure, both for sister chromatid cohesion and for
chromosome condensation (Hartman et. al. JCB 151: 613, 2000, Panizza et.al Curr. Biol.
10:1557, 2000). Homologous genes have been found in as diverse species as Neurospora
and humans (Sumara et. al. JCB 151:749, 2000). One of the best characterized
homologues is SPO76 from Sordaria macrospora (van Heemst et.al. Cell 98: 261, 1999).
SPO76 localizes to mitotic and meiotic chromosomes and has been shown to play a
critical role at numerous intervals during chromosome morphogenesis. In particular the
spo76-1 allele has significant defects in meiotic chromosome development leading to a
failure to complete meiosis and produce viable spores. We wished to further investigate
the role of this gene in meiosis in S.cerevisiae. We are studying the timing and
chromosomal localization of the wild type protein using GFP tagged constructs. In
addition we are looking into PDS5p’s interactions and colocalization with other meiotic
proteins involved in sister chromatid cohesion
33
C2.53 - The Arabidopsis SWI1 protein is required for both chromatid arm and
centromere cohesion during meiosis
R. Mercier, D. Vezon, E. Bullier, G. Pelletier, C. Horlow, INRA, Versailles, France
Many plant mutants affected in meiosis have been described but only a few genes have
been cloned and characterised. Swi1, an Arabidopsis T-DNA tagged mutant was obtained
in our group and previously described as affected exclusively in female meiosis
(Motamayor et al 2000, Sex. Plant. Reprod. 12:209-218 ).
A second allele (swi1.2), which fails to complement swi1.1, was isolated from a
chemical mutagenised population. Swi1.2 is completely sterile and is affected in both
male and female meiosis. Investigation of meiotic behaviour shows a highly unusual
phenotype. The classical steps of the meiotic prophase are completely abolished and
chromatid arms and centromeres loss their cohesion in a stepwise manner before
metaphase I. Therefore 20 chromatids instead of 5 bivalents are present at the metaphase
plate and proceed to an aberrant segregation.
The wild type phenotype was restored in the two mutants by insertion of a 5 kb
genomic fragment, allowing us to define the SWI1 gene. The encoded protein does not
show strong similarity with any known proteins from plant or other organism. SWI1-GFP
fusion shows that SWI1 protein is present in male and female meiocyte nuclei, at premeiosis and at very early prophase stage.
SWI1 is a good candidate for a new kind of protein involved in chromosome
structure and chromatid cohesion establishment during meiosis.
C2.54 – Analysis of the pleiotropic phenotype resulting from RNA interference of
Rad-51 in C. elegans
C. Rinaldo, P. Bazzicalupo, M. Hillard and A. La Volpe, International Institute of Genetics and
Biophysics, Naples, Italy, S. Ederle, Universitá di Napoli, Naples, Italy
C2.55 - A possible link between meiotic histone phosphorylation, kinetochore-site
selection and selective loss of sister chromatid cohesion in C. elegans
P. Pasierbek, M. Jantsch, D. Schweizer and J. Loidl, Institute of Botany, University of Vienna,
Austria
The chromosomes of the nematode C. elegans are holokinetic in mitosis, whereas in both
meiotic divisions centromeres are localized [Albertson &Thomson 1993. Chromosome
34
Res. 1: 15-26]. It is not yet known whether these facultative centromeres occupy the
chromosome ends or an extended tract along the chromosome arms. We have shown that
reductional and equational meiotic divisions are staged by the loss of sister chromatid
cohesion in the distal part (beyond the chiasma) of bivalents at the first meiotic division,
and in proximal regions at the second meiotic division [Pasierbek et al., in press]. We
performed double immunostaining with antibodies against REC-8, a meiotic cohesion
protein, and phosphorylated (Ser10) histone H3 (PhoH3) and found the association of
PhoH3 with the short axes of cross-shaped diakinesis/metaphase I bivalents. This
distribution is in agreement with two favoured non mutually exclusive interpretations: 1)
PhoH3 protects the distal portions of arms from microtubule attachment, confining the
functional kinetochore to the region from one chromosome end to the (mostly single)
chiasma; 2) PhoH3 marks the chromosomal regions where sister chromatid cohesion is to
be released at the onset of anaphase I in order to allow the disjunction of homologs. This
notion is supported by observations that H3 phosphorylation is effected by AIR-2 kinase
and that AIR-2 –depletion causes meiotic nondisjunction [Hsu et al. 2000. Cell 102: 279291; Kaitna et al. 2000. Curr. Biol. 10: 1172-1181, and lit. cit. therein].
C2.56 - Sister chromatid axis assembly in mammalian meiotic cells
J. Pelttari, M.-R. Hoja, L. Yuan, J.G. Liu and C. Höög, Department of Cell and Molecular
Biology, Karolinska Institutet, Sweden
The synaptonemal complex is a meiosis-specific protein complex that is important for
pairing, synapsis, recombination and segregation of homologous chromosomes.
Ultrastructural analysis of the SC reveals a tripartite structure with two parallel lateral
elements (LE) and a central element (CE). The LEs, holding the sister chromatids of each
homologous chromosome, and the CE are held together by the transverse filaments (TF).
Three genes encoding SC proteins in mammals have been identified, SCP1 encoding a
component of the TF and SCP2 and SCP3 encoding components of the LE. The main
interest in this study is the search for protein interactions with or within the synaptonemal
complex. We know that there are several meiotic proteins that localize along the LEs and
are potential candidates for interacting with the LE-proteins. Recently, it has been shown
that the cohesin proteins, Smc1 and Smc3, have a role in meiosis. As in mitotic cells, the
cohesins are important for sister chromatid cohesion. In SCP3 knockout experiments, we
have the unique possibility to see if and how the lack of SCP3 influences other proteins.
All of the proteins known to localize to the SC, for example the cohesins, and the
recombination proteins, Rad51 and Dmc1, are potential candidates for interacting with
the SC-proteins. In addition, in overexpressed somatic cells, we can look for
colocalization, that may suggest an interaction between the analyzed proteins.
35
C2.57 - The analysis of x-, y-chromosome pairing in some rodents from the genera
Microtus and Apodemus
L.D. Safronova*, M.I. Baskevich*, V.M. Malygin**
* Severtsov Institute of Ecology and Evolution, Moscow; ** Moscow State University, Moscow; Russia
The meiotic (EM SK analysis of meiotic prophase - stage pahytene) and mitotic (G-,Cbanding) chromosomes of Microtus arvalis, M. rossiameridionalis, M. (T.) daghestanicus,
Apodemus peninsular, A. flavicolis, A. ponticus and A. uralensis have been studied. The
focus of attention on X-, Y-chromosomes pattern pairing was made. Pairing of X- and Y
chromosomes at meiotic prophase in species under study was analysed in relation to the
taxonomic position of species under study and variation in the morphology of their sex
chromosomes. The asynaptic behaviour of X-, Y-chromosomes in meiotic prophase I in
Microtus has been marked. On the contrary representatives of Apodemus under study
demonstrated synapsis of X-, Y-chromosomes in meiotic prophase I which depends on
the substage of pahytene and species rank. The possible relationships among the sex
chromosomes behaviour and heterochromatin pattern of X-, Y-chromosomes and their
morphology are discussed. The hypothesis of mammalian heterochromosomes evolution
on the examples of Microtus and Apodemus genera are estimated.
C2.58 - Sister chromatid cohesion proteins in mammalian meiosis
M. Eijpe1, H.H. Offenberg1, E. Revenkova2, R. Jessberger2 and C. Heyting1
1
Laboratory of Genetics, Wageningen University, 2Institute for Gene Therapy and Molecular Medicine,
Mount Sinai School of Medicine, New York
Structural maintenance of chromosomes (SMC) proteins fulfil pivotal roles in
chromosome dynamics and metabolism. In yeast, the SMC1/SMC3 heterodimer is
required for mitotic and meiotic sister chromatid cohesion, and for DNA recombination.
Little is known, however, on mammalian SMC proteins in meiotic cells. We have
identified a novel, meiosis-specific SMC protein named SMC1 which, except a unique
highly basic C-terminal domain, is around 70 % homologous to SMC1 (SMC1ß). The
gene is specifically expressed in testis, and the SMC1ß protein co-immunoprecipitated
with SMC3 from testis nuclear extracts, but not from a variety of somatic cells. Analysis
of testis sections and chromosome spreads of various stages of meiosis revealed specific
expression of SMC1ß in meiosis I and II. SMC1ß localizes along the axial elements of
synaptonemal complexes (SCs) in prophase I, and is present in preparations of SCs.
Some SMC1ß but no SMC1ß remains chromatin-associated near the centromeres up to
metaphase II. Thus, SMC1ß and not SMC1ß is likely involved in maintaining cohesion
between centromeres until anaphase II. We also present the immunolocalisation of other
proteins involved in sister chromatid cohesion in meiosis.
36
C2.59 - Ultrastructural transformation of the lateral elements of synaptonemal
complexes at the diplotene stage of meiosis in rye and mouse
Y.F.Bogdanov, O.L. Kolomiets and Y.S. Fedotova, The Vavilov Institute of General Genetics,
Moscow, Russia
As it is known for plant meiosis, discontinuous SCs transform during the diplotene stage
into the linear arrangements (sequences) of SC stretches with gaps in between. The
picture is typical for microspreads routinely stained with AgNO3 solution, pH 6.0-7.0.
After we stained similar microspreads of rye meiocytes with AgNO3 solution, pH 3.54.5, we found that the gaps contained unpaired (desynapsed) SC lateral elements (LEs)
which, at this stage, were expanded into threads and repulsed from each another. They
formed lateral loops in early diplotene, and irregular coils in late diplotene. Thus our
early observation [1] is now extended. We arrive at the conclusion that, during the
diplotene stage, SCs degrade stepwise into the linear arrangements of SC stretches
discontinuously connected with thin threads of unpaired and transformed axial cores
(former LEs), each core being dissociated into two sister threads. In the course of the
diplotene stage, SC segments become shorter and unpaired axial cores become longer.
In mouse diplotene spermatocytes, desynapsed LE transforms into two expanded wavy
axial cores, which connect short SC stretches. Taking into account that mammalian LEs
consist of, at least, two proteins, SCP2 and SCP3, which possess some DNA-protein and
protein-protein interactions, several models of their intermolecular interactions and
transformations during the diplotene stage could be considered.
1. Fedotova, Y.S., Kolomiets, O.L. & Bogdanov Y.F. (1989) Genome 32: 816-823.
C2.60 - Analysis of chiasma frequency in Arabidopsis thaliana accession Wassileskija
and in two meiotic mutants
E. Sanchez Moran, J.L. Santos, Departamento de Genetica, Universidad Complutense de Madrid,
Madrid 28040 Spain; S.J. Armstrong, F.C.H. Franklin, G.H. Jones, School of Biosciences, The
University of Birmingham, Birmingham B15 2TT UK
Meiotic chiasmata were analysed in metaphase I pollen mother cells (PMCs) of wild-type
Arabidopsis thaliana and in two meiotic mutants. Fluorescence in situ hybridisation
(FISH) with 45S rDNA and 5S rDNA as probes was used to identify the five
chromosome pairs.A wild-type chiasma frequency of 9.24 per cell was found, consistent
with estimated genetical recombination values. Individual bivalent chiasma frequencies
varied according to chromosome size; chromosome 1 had the highest mean chiasma
frequency (2.14) while the short acrocentric chromosomes had the lowest frequencies
(1.54 and 1.56). FISH analysis was extended to two meiotic mutants (asy1 and dsy1)
having low residual bivalent and chiasma frequencies. Mutant dsy1 gave no indication of
chromosome preference for residual bivalent formation; instead it showed a general
reduction in bivalent and chiasma frequencies. In asy1 the longest chromosome (1) had
the lowest bivalent frequency and chiasma frequency while the short acrocentric
37
chromosome 2 had the highest frequencies. This chromosome pair may be preferentially
involved in synapsis and chiasma formation because of their association with the
nucleolus. However, other factors may be operating since the other acrocentric
chromosome (4), with similar size and structure to chromosome 2, did not share these
chiasma properties.
C2.61 - Asy1 localizes to meiotic chromosomes at early prophase of Arabidopsis
meiosis
A.P. Caryl, S.J. Armstrong, G.H. Jones and F.C.H. Franklin, University of Birmingham, UK
One of the key events of meiosis is homologous chromosome synapsis. We have
identified an Arabidopsis mutant, asy1, which undergoes a primary failure of synapsis
during both male and female meiosis. This leads to univalent formation at the first
meiotic division, and a reduced fertility phenotype. The Asy1 gene has been cloned and
asy1 has been shown to be a null mutant. The N-terminal region of the Asy1 protein
exhibits similarity to the N-terminal region of the Hop1 protein of Saccharomyces
cerevisiae. The Hop1 protein is known to be important in synaptonemal complex
assembly, normal synapsis and crossing over. In order to determine where the Asy1
protein is localized during Arabidopsis meiosis recombinant protein was expressed in E.
coli and used to produce polyclonal antiserum. Preliminary western blot experiments
have shown that the antiserum is specific, recognizing a single band present only in
meiotic tissue. Preliminary imunolocalization has shown that the Asy1 is localized to the
chromosomes of meiotic cells during zygotene/pachytene. No Asy1 protein was detected
in later meiotic stages or in the surrounding somatic cells. The antiserum also recognizes
a potential Asy1 homologue on western blots of meiotic tissue from Brassica oleracea.
Imunolocalization experiments show that this protein has a similar meiotic expression
pattern to Asy1 in Arabidopsis.
Caryl et al (2000), Chromosoma, 109 (1-2): 62-71
C2.62 - Meiosis in Arabidopsis
L.A. Sherratt, A. Beven, J. Doonan, G. Moore and P. Shaw, John Innes Centre, UK
Arabidopsis has proved to be an excellent model organism in plant biology, due to its
short life cycle, small nuclear genome, and well-established genetic and molecular
methods. However, from a cytological viewpoint, it is difficult to work with because of
its small size. Meiosis in particular is problematic, because the process is relatively rapid
and the floral buds are small. To address this problem two approaches are being used in
Arabidopsis. The behaviour of centromeres and telomeres are being observed by
fluorescent in situ hybridisation on floral bud sections, which enable positional
38
information to be retained. Previous work on wheat and other cereals has shown that
centromere association prior to meiosis occurs in the polyploids, but not in the diploids.
To see whether this extends outside of the cereals, we are now carrying out a similar
analysis on diploid and tetraploid Arabidopsis. In the second approach, individual
chromosomes of Arabidopsis are being tagged with green fluorescent protein (GFP). A
number of distinct lines with tags on various chromosomes and at different locations are
being generated, enabling direct visualisation of meiosis and, in particular, homologue
pairing. The meiocytes can then be analysed by 3D optical imaging technology. GFP
analysis, coupled with the in situ experiments examining the general organisation of the
chromosomes in meiocytes, is a powerful method for studying meiosis in plants.
C2.63 - Tracing of Arabidopsis chromosome 4 during meiosis by chromosome
painting
M.A. Lysak1, P.F. Fransz2, H.B.M. Ali1, S. J. Armstrong3, G.H. Jones3, I. Schubert1
1
Institute for Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany; 2 Swammerdam
Institute for Life Sciences, Amsterdam, The Netherlands; 3 University of Birmingham, Birmingham, UK
Studying chromosome dynamics during meiosis is often hampered by the difficulty to
identify and trace an entire chromosome, especially at early meiotic stages. Here we
report on a new approach to monitor individual Arabidopsis chromosomes during
subsequent meiotic stages. BAC clones covering the entire chromosome 4 of Arabidopsis
thaliana were individually labelled and simultaneously hybridized to meiotic
chromosomes and premeiotic interphase nuclei using fluorescent in situ hybridization
(FISH). In total 139 BAC DNA clones, 26 from the short arm and 113 from the long arm,
were used. This resulted in an alternating red and green fluorescent pattern. By carefully
selecting the label for each BAC or group of BACs, particular regions could be identified.
Cross-hybridization to remaining chromosomes of the Arabidopsis complement was
negligible. The obtained results represent a significant achievement in plant cytogenetics
as an entire chromosome was successfully painted for the first time in an euploid plant
species.
Tracing of the entire chromosome 4 was most easy in pachytene cells, when the
homologs are paired forming a bivalent structure. However, also at early meiotic stages,
leptotene and zygotene, pairing and/or alignment behaviour of homologs could be
examined. Moreover, this painting approach enabled us to analyse initiation sites for the
alignment and pairing at early prophase I. In conclusion, chromosome painting is
demonstrated as a powerful technique for analysis of chromosome dynamics during
meiosis in Arabidopsis.
39
C2.64 - Tracking the bouquet and detecting Rad51/Dmc1 in meiosis of rye (Secale
cereale L.)
E.I. Mikhailova, O.A. Tikholoz and S.P. Sosnikhina, Genetics, State University, St.Petersburg,
Russia; H.H. Offenberg and C.Heyting, Genetics, Research University, Wageningen, The
Netherlands; R.N. Jones and G. Jenkins, The University of Wales, Aberystwyth, UK.
The nuclear disposition of pericentromeric and subtelomeric domains revealed by FISH
was compared with the distribution of the immunocytologically detected recombinogenic
protein Rad51/Dmc1 in pollen mother cells (PMCs) at pre-meiotic interphase and
prophase I in wild type (WT) and in two asynaptic mutants. Homozygotes for the sy1 and
sy9 non-allelic mutations form axial elements during leptotene of male meiosis, but fail to
form synaptonemal complexes. Consequently, recombination is severely impaired, and
high univalency is observed at metaphase I. Simultaneous FISH with pSc200 and CCS1
probes reveals clusters of subtelomeric and pericentromeric domains opposing each other
in the nuclei at the onset of leptotene in PMCs of WT and sy9 mutant. By contrast, in sy1
two to five aggregates of subtelomeric domains are formed which fail to disperse at later
stages of meiotic prophase, while pericentromeric domains are dispersed prematurely in
the majority of the nuclei. Immunostaining of a Rad51/Dmc1 homolog, by means of a
polyclonal antibody raised to full-length tomato Rad51 protein [Anderson et al., 1997],
reveals clusters of foci at zygotene which co-localise with the subtelomere clusters in WT
and sy9 mutant. By contrast, there is almost no signal at leptotene-zygotene of sy1
mutant. It is supposed that sy1 mutation disrupts the nuclear disposition of centromeres
and telomeres at the end of pre-meiotic interphase, while absence of Rad51/Dmc1 foci at
prophase I indicates that also recombination is affected by this mutation. The differences
between two mutants will be discussed in terms of the relationship between bouquet
dynamics, synapsis and recombination.
C2.65 - Meiosis and organization of repeated sequences in rye and wheat
Trude Schwarzacher, Department of Biology, University of Leicester, UK, Tatanja Garkoucha,
John Innes Centre, Norwich, UK, Angelines Cuadrado, University of Alcala, de Henares, Madrid,
Spain
During chromosome pairing and recombination, there are multiple interactions between
genomes, chromosome, DNA sequences and nucleotides that are unique to the early
stages of meiosis. Using molecular cytogenetics methods, we have examined the
behaviour of chromosomes and the physical organization of specific DNA sequences
during meiosis. Simple sequence repeats (SSRs)- short DNA motifs such as (CAA) and
(CAGA) – are abundant in the plant genomes and form major blocks of repetitive DNA
in rye and wheat. At leptotene to pachytene, SSR clusters occur mainly in the chromatin
loops formed along the synaptonemal complex, while subtelomeric long satellite
sequence families are closely associated with the complex, showing the specificity of SCDNA interactions and the possible role of different sequences classes. The appearance
and localization of structural and enzymatic proteins involved in breakage and rejoining
40
of DNA molecules and promoting synapsis can be followed. Comparison of genetical and
physical maps reveals that gene are clustered genetically near the centromere while they
are physically near the telomere. The increased number of subterminal chiasmata is
reflected by an increased pachytene lengths, but not synaptonemal complex length. Foci
of DMCI and RAD51 show remarkable clustering at the ends of zygotene chromosome,
indicating that initiation rather than maturation of recombination events is responsible for
the non-random distribution of chiasmata.
C2.66 - The roles of Tid1/Rdh54 and Dmc1 in crossover control during meiotic
recombination
Miki Shinohara 1, 2, Doug Bishop 2 and Akira Shinohara 1,2
1
Dept. of Biology, Graduate School of Science, Osaka University, Osaka 560-0043, Japan, 2 Dept. of
Radiation and Cellular Oncology, University of Chicago, IL 60637, USA
Crossover recombination ensures disjunction of homologous chromosomes during
meiotic division I. The distribution of crossover is strictly controlled so that one
homolog pair has at least one crossover. A crossover at one site lowers the possibility of
the second crossover nearby on chromosomes. This crossover interference is one of
manifestation of crossover control.
Crossover interference operates in cis on
chromosomes in a range of more than 100kb in yeast and more than 1MB in human.
Two RecA homologues, Rad51 and meiosis-specific Dmc1, play a role in the
formation of crossover between homologous chromosomes. Rad51 and Dmc1 work
together with Rad54 and a RAD54 homologue, Tid1/Rdh54, to promote recombination
between homologous chromosomes. Particularly, TID1/RDH54 is necessary for the coassociation of Rad51 and Dmc1 on meiotic chromosomes.
Here we show that mutants in the TID1lRDH54 and DMC1 genes are defective in
crossover interference without affecting crossover frequencies. We propose that the
cooperation of two RecA homlogues, Rad51 and Dmc1, plays a critical role in the
crossover control during meiosis.
C2.67 - The WWMeB: a germline gateway
R.E. Esposito (University of Chicago, USA), M. Primig, J. Demaille, N. Lamb, C. Sarrauste
de Menthière (IGH, CNRS, Montpellier, France), H. Dickinson (Oxford University, UK), M.
Fellows (Institut Pasteur, Paris, France), A. Grootegoed (Erasmus University, Rotterdam, The
Netherlands), R.S. Hawley (University of California, Davis, USA), P.A. Hunt (Case Western Reserve
University, Cleveland, USA), B. Jegou (GERM, INSERM, Rennes, France), B. Maro (Universite Paris
VI, France), A. Nicolas (Institut Curie, Paris, France), T. Orr-Weaver (MIT, Cambridge, USA), T.
Schedl (Washington University, St. Louis, USA), A. Villeneuve (Stanford University, USA), D.J.
41
Wolgemuth (Columbia University, New York, USA), M. Yamamoto (University of Tokyo, Japan),
D. Zickler (Universite Paris-Sud, France)
The World Wide Meiosis dataBase (WWMeB) is a gateway of gametogenesis. It is the
first subject-oriented database that covers a fundamentally important biological question
studied in many model systems. The WWMeB’s goals are to provide a rapid access to a
comprehensive compilation of genes, expression data and functions implicated in
germline development, meiosis, gamete formation, and gamete function in various key
model systems. The WWMeB enables the scientific community studying these processes
to directly contribute and update such information after review by an international board
of curators to ensure the highest possible scientific quality of the databases content. The
principle of the WWMeB is applicable to all fields of biological research that investigate
important, evolutionarily conserved phenomena.
C2.68 - A search for somatic chromosome pairing in Saccharomyces cerevisiae
A. Lorenz, J. Fuchs, R. Buerger and J. Loidl, Institutes of Botany and Mathematics, University of
Vienna, Austria
The existence of somatic chromosome pairing, whilst well-known from dipterans, is still
under debate in other organisms. A moderate level of homologous associations was
reported to occur in diploid strains of the budding yeast [Burgess et al. 1999. Genes Dev.
13: 1627-1641; Burgess & Kleckner 1999. Genes Dev. 13: 1871-1883], with possible
functions in the trans-regulation of transcriptional activity (transvection) and
recombinational repair of DNA lesions. We have compared in various strains intranuclear
distances between differently coloured FISH markers at homologous and nonhomologous chromosomal loci. Probes from corresponding chromosome regions with
respect to the centromere were used to compensate for the influence of the parallel
polarized arrangement of chromosome arms (Rabl-orientation) on the neighbourhood of
homologous loci [Jin et al. 2000. J.Cell Sci. 113: 1903-1912]. We have also simulated the
expected random distribution of chromosomes in spread yeast nuclei by a computer
model and asked if there is a deviation of homologous chromosomes towards a closerthan-random distribution. By means of these simulations we could uncover several
experimental parameters which can lead to a bias towards pairing. Considering these
sources of error, the observed that the distribution of homologs closely resembles the
computer-simulated random distribution. In general, there is only little evidence for
somatic pairing. However, there is some indication that certain chromosomal loci may
show a slight tendency for homologous interactions and that certain stress conditions, like
irradiation, lead to a spatial reorganization of nuclei.
C2.69 - The human meiosis-specific MutS Homolog, hMSH4, interacts with PMS2
42
S. Santucci-Darmanin, F. Lespinasse and V. Paquis-Flucklinger, UMR CNRS/UNSA 6549,
Faculté de Médecine de Nice (France)
MSH4 is a meiosis-specific MutS Homolog. In S. cerevisiae and C. elegans, this protein
is required for reciprocal recombination and proper segregation of homologous
chromosomes during meiosis. PMS2 is a MutL homolog. Analysis of male PMS2deficient mice has revealed that PMS2 is involved in synapsis of homologous
chromosomes during mammalian meiosis. Here, we show that the human MSH4
(hMSH4) protein interacts with PMS2 and that this interaction does not require the
binding of hMSH4 to DNA. These results suggest that these two proteins are involved in
a common pathway during mammalian meiosis. We have recently shown that hMSH4 is
present at sites along the synaptonemal complex as soon as homologous chromosomes
synapse. Others have shown that hMSH4 is required for chromosomes synapsis in
mammals. Taken together all these results suggest that, in mammals, MSH4 is involved
in chromosome synapsis in conjunction with PMS2. We have previously observed that
MSH4 interacts also with the MutL Homolog 1 (MLH1), a protein that is not involved in
synapsis but which is essential for crossing-over both in yeast and mammals. Moreover,
hMSH4 co-localizes with MLH1 on chromosomes at the early-mid pachytene stage of
meiotic prophase. These results indicate that, in mammals, MSH4 acts in conjunction
with MLH1 to promote crossing-over. Taking into account all these informations, we can
assume that, in mammals, MSH4 is first required for chromosome synapsis and that this
protein is involved later in crossing-over events. Our results indicate that at these two
steps, MSH4 acts with different MutL homologs.
C2.70 - Regulation of expression and function of Cyclin A1 during meiosis in
mammalian germ cells
K. Lele, D. Liu, C. Liao, X. Wang, and D.J. Wolgemuth*, Genetics and Development,
Columbia University College of Physicians and Surgeons, New York, USA
The two mammalian A-type cyclins have strikingly different expression patterns in both
mice and humans. In mice, the cyclin A2 gene (Ccna2) is expressed widely, while the
gene for cyclin A1 (Ccna1) is expressed exclusively in male germ cells at a stage of
development that Ccna2 is not present. Ccna1 expression appears in spermatocytes
during late prophase through the first meiotic cell division (MI), suggesting its function at
the G2/M transition of this unique cell division. Targeted deletion of Ccna1 in mice
produced a complete block in spermatogenesis prior to MI. Meiotic arrest was
accompanied by a defect in MPF (M-phase-promoting factor) activity at the G2-M
transition of MI, suggesting that Ccna1 may act upstream to promote this transition.
Ccna1 mRNA and protein co-localize in spermatocytes, suggesting that Ccna1 is
regulated at the level of transcription. We have used both sequence analysis and a
transgenic approach to study mechanisms of expression and repression of Ccna1 in vivo.
Approximately 8 kb or 5 kb of Ccna1 upstream flanking sequence appears sufficient to
direct expression of a lacZ reporter gene to late prophase spermatocytes and to maintain
repression of expression in other tissues. Approximately 1 kb of upstream flanking
43
sequence also appears to direct expression of lacZ to spermatocytes, but variably. In
addition, ectopic expression was detected in other types of tissues. The flanking
sequence between 5 kb and 1 kb contains consensus binding sites for transcription factors
that appear to be co-expressed with Ccna1 in spermatocytes, including Myb, NF-KB, and
YY1.
C2.71 - Kar3p is not required for bouquet formation in haploid yeast meiosis
H. Scherthan1, J. Loidl2 and E. Trelles-Sticken1
1
Univ. of Kaiserslautern, Ger.; 2Univ. of Vienna, Austria
The lengthy prophase of Meiosis I is characterized by extensive chromosomal
movements which culminate in the tight clustering of telomeres at the leptotene-zygotene
transition. Recently, we have shown that deletion of a meiosis-specific telomere protein
of S. cerevisiae, Ndj1p, results in bouquet disruption and leads to inefficient and retarded
and disordered meiotic homologue pairing [Trelles-Sticken et al. 2000. J.Cell Biol.
151:95-106]. Furthermore, we observed that bouquet formation is independent of
recombination and does not require homologues, since it occurred in a meiosis-competent
haploid yeast strain. Another candidate protein thought to be responsible for bouquet
formation is the kinesin-like motor protein Kar3p [Bascom-Slack and Dawson. 1997. J.
Cell Biol. 139:459-467]. This prompted us to determine the effect of a KAR3 deletion on
telomere clustering in a meiosis-competent haploid yeast strain. To be able to distinguish
meiocytes from non-meiotic cells we expressed a HA-epitope-tagged version of the
meiosis-specific telomere protein Ndj1 in our haploid strains. Bouquet formation and the
degree of induction of the meiotic cycle was then determined by telomere- and
centromere-FISH and by Ndj1-IF to meiocytes from synchronized mutant and control
cultures. Surprisingly, we detected bouquet formation in both the control and the kar3∆strain, which suggests that Kar3p is not a bouquet motor. However, we also observed that
the induction of meiosis is strongly reduced in kar3 as compared to the control KAR3
strain, which suggests that Kar3p may be important for other than bouquet functions possibly in the premeiotic division.
C2.72 - Atce1, a novel CREB family member specifically expressed in post meiotic
spermatids
Gil Stelzer and Jeremy Don, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
Evolutionary conservation of the various meiotic stages could reflect functional
evolutionary conservation of genes taking part in the process. Tctex2, a previously
identified murine gene, was recovered from a functional screen for mouse genes that can
activate yeast early meiotic promoters. In an effort to identify mouse proteins that
44
interact with Tctex2, a mouse testis expression library was screened via the Two Hybrid
System. Subsequently, a clone consisting of two reading frames, with a frameshift of one
nucleotide, was isolated. The peptide encoded by one of the reading frames interacted
with Tctex2. However, the other reading frame encoded a 315 amino acid peptide with
sequence homology to several transcription factors, especially to CREB3 of the
CREB/ATF family. CREB (CRE Binding protein) proteins bind a specific DNA
sequence called CRE (cAMP Responsive Element) and are activated by PKA
phosphorylation. The new CREB like peptide, designated Atce1, consists of discrete
functional domains, including - DNA binding and leucine zipper homodimerization
domains. Northern blot analysis revealed that Atce1 is specifically expressed in the testis.
A developmental study showed that transcripts begin accumulating in post natal day 21
testes culminating in day 24, reflecting expression in round and elongating spermatids.
This was confirmed by In situ hybridization analysis. The structural similarity between
Atce1 and the CREB group of genes, coupled with the preliminary expression pattern
suggest the possibility that the Atce1 protein binds CRE sequences of late spermatogenic
genes and acts as a transcription activator or alternatively as a transcriptional repressor of
these genes.
C2.73 - Synaptonemal complexes of zebrafish
B.M.N. Wallace and H. Wallace, School of Biosciences, University of Birmingham, U.K.
We have obtained surface-spread preparations of synaptonemal complexes from both
sexes of the zebrafish, Danio rerio. We used ovaries from young females, 25-30 mm
long, before most oocytes had entered the growth phase. Testes were taken from young
adult males with relatively few mature sperm. The general speading technique has been
described (Wallace et al., 1992).
Chromosome pairing begins at the telomeres. Both oocytes and spermatocytes show
conventional complete pairing of homologues at pachytene. Centromeres are only
detectable in some preparations, but should allow us to prepare an SC karyotype. Our
observations conform to prior evidence that zebrafish probably do not have sex
chromosomes. 25 synaptonemal complexes are evident at pachytene, with no partially
paired configuration which might be taken to identify a sex bivalent.
B.M.N. Wallace, J.B. Searle & C.A. Everett. Cytogenet. Cell Genet. 61:211-220 (1992).
C2.74 - Cytological analysis of the effects of Ph1 and Ph2 loci of polyploid wheat by
means of GISH procedure
E. Sánchez-Morán, Departamento de Genética, Facultad de Biología, 28040 Madrid, Spain
45
The effect of different mutants, ph1b, ph1c, ph2, involved in the regulation of
chromosome pairing in polyploid wheats has been analyzed by means of a genomic in
situ hybridization method that allowed us the unequivocal identification of A, B and D
wheat genomes. All plants of allohexaploid wheat (AABBDD) carrying the ph1b
mutation showed changes in the structure of the chromosomes and some of them also in
the number of chromosomes. Chromosome rearrangements between A and D genomes
were predominant. However, a total absence of rearrangements were observed both in
plants of durum wheat (AABB) carrying the ph1c mutation and in AABBDD plants
carrying the ph2 mutation. Taking into account these findings, the possible effect of Ph1
and Ph2 loci on meiotic pairing are discussed.
C2.75 - Targeted mutagenesis of DMC1
T. M. Holzen, Committee on Genetics, University of Chicago, Chicago, Illinois, USA; J. M.
Logsdon, Jr., Department of Biology, Emory University, Atlanta, Georgia, USA; D. K. Bishop,
Department of Radiation and Cellular Oncology, University of Chicago, Chicago, Illinois, USA
In S. cerevisiae, the RecA homologs DMC1 and RAD51 are required for wild type levels
of meiotic recombination. Although these two gene products are thought to share strand
exchange activity, recent data have shown they also have separate functions during
meiosis. For example, Dmc1 promotes recombination in the context of normal meiosisspecific structures that repress Rad51. In an attempt to disrupt Dmc1-specific function,
we are mutagenizing amino acid residues that are highly conserved among DMC1
orthologs but not among RAD51. Here we report the identification of two DMC1
mutants, dmc1F222A and dmc1N254A that exhibit null dmc1 phenotypes for sporulation
and spore viability. A third mutant, dmc1V224A, displays a delay in sporulation as well
as decreased sporulation. Mutants in which V224 has been converted to threonine, the
corresponding residue in RAD51, also exhibit this mutant sporulation phenotype.
Surprisingly, dmc1V224A mutants exhibit a lower spore viability in a spo13 background,
in which chromosomes tend to segregate equationally during the first and single meiotic
division. These preliminary data suggest that DMC1 interacts with SPO13 in an
unexpected manner. We hope further characterization of these mutants as well as other
mutants isolated from the screen will identify residues and regions of DMC1 and
molecular interactions employed by Dmc1 that confer its specialized functions.
C2.76 - Recombination dynamics of subtelomeric loci in S. cerevisiae
Craig D. Griffin and E. J. Louis, University of Leicester, Department of Genetics, University Rd,
LE1 7RH
In the budding yeast S. cerevisiae locus chromosomal position influences recombination
dynamics. For example, telomere proximal loci (within 30kb of the telomere sequences
46
are typically considered subtelomeric) have reduced allelic, meiotic recombination
compared to chromosome-internal/interstitial loci, as has been shown using microarray
and genetic methods. Using two non-functional mutant heteroalelles of nutritional
markers (egg. URA3 and LYS2) we are further characterising recombination of telomere
proximal sequences. Previous observations indicate, meiotically and mitotically,
subtelomeric sequences recombine less frequently with homologous-internal sequences
on heterologous chromosomes than with other subtelomeric sequences (and vice-versa).
The working title of this phenomenon is Recombination Barrier and reverse genetic
screens are being used to identify potential genetic factors involved. The strains have
been engineered that shall be used to delineate candidate cis-acting sequences of the
mitotic and meiotic barriers. At present all data suggests the mitotic barrier to be a
consequence of the physical sequestration of telomeres at the interphase nuclear
periphery while the meiotic barrier appears to be a function of the temporal isolation of
subtelomeric (late S-phase) sequence replication from that of interstitial (early S-phase)
loci.
C2.77 - The unusual chromatin structure of the central domain of
Schizosaccharomyces pombe centromeres is maintained during meiosis
Julia Smirnova and Ramsay McFarlane, Cell & Molecular Biology Group, School of Biological
Sciences, University of Wales – Bangor, Memorial Building, Deiniol Road, Bangor, Gwynedd, LL57
2UW, United Kingdom
The transition from the mitotic to the meiotic cell cycle requires reconfiguration of the
centromeres to ensure sister chromatids migrate to the same pole during anaphase I.
Recent work in the fission yeast, S.pombe, has shown that Rec8p is required for correct
centromere reconfiguration during pre-meiotic DNA replication to enable reductional
division at meiosis I.
S.pombe has relatively large complex centromeres. During mitotic proliferation
the central domains of the centromeres have an unusual chromatin structure that renders
them more labile in the presence of microccocal nuclease than bulk chromatin. The
formation and maintenance of this chromatin structure during mitosis is dependent upon
the function of at least three known proteins, Mis6p, Mis12p and Cnp1p (the S.pombe
CENP-A homologue).
In this study we have analysed the S.pombe centromere central domain chromatin
structure during a highly synchronised meiosis. We find that despite the altered function
of the centromeres, the unusual central domain chromatin structure is maintained
throughout meiosis. These findings will enable us to determine whether or not the
maintenance of this unusual chromatin structure is dependent of the same factors
controlling mitotic central domain structure, or whether there are novel factors
functioning during the meiotic cell cycle.
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C2.78 - Pairing of terminally deficient chromosomes in wheat
Pete Carlton(1), Bernd Friebe(2), Bikram Gill(2) and W. Zacheus Cande(1)
(1) University of California at Berkeley, MCB Dept. (2) Kansas State University, Dept. of Plant Pathology
A rye deletion series in a wheat background (Chinese spring) was used to observe the
meiotic pairing behavior of a single pair of chromosomes, and to assess the effects of
terminal deficiencies on the process of meiotic pairing. Preliminary observations on latestage meiotic material have shown that homozygous deficiencies have little or no effect
on pairing, while heterozygous deficiencies can lead to severely reduced chiasma
formation. While the effect is striking, little is known about how it is brought about, as
conventional techniques can only observe chromosomes that have already condensed
sufficiently to be visible. The current work uses three-dimensional fluorescent in situ
hybridization to highlight the rye chromatin against the wheat background, so that earlier
meiotic stages can be observed. The goals of the project are: first, to characterize the
behavior of non-deleted chromosomes with particular attention to telomere and
centromere behavior; and secondly, to ascertain how heterozygous deficiency is able to
interfere with pairing by noting when and where deviations from normal behavior are
occuring. Observations so far have suggested that terminal deficiency causes
mislocalization of a chromosome end during the bouquet stage, away from the telomere
cluster and into the nuclear space.
C2.79 - Simulation and time-course analysis of bouquet formation
Pete Carlton(1), Carrie Cowan(2) and W. Zacheus Cande(1,2)
(1) University of California at Berkeley, MCB Dept. (2) University of California at Berkeley, Dept. of
Plant and Microbial Biology
The bouquet, or clustering of chromosome ends in a small region of the nuclear envelope
during meiotic prophase, has been proposed for many functions such as promoting the
alignment of homologous chromosomes and providing a physical anchor for models of
interference. Though mutants that interfere with its formation are known, the mechanism
of bouquet formation is still poorly understood. As it has not been observed in the
process of formation in any organism, few if any constraints on mechanism are known.
To address this problem, we are comparing computer models of bouquet formation with
data taken from time course experiments in rye, to narrow down the hypotheses. The
models are implemented as kinematic polymers (chromosomes) diffusing in a confined
space. Though several distinct mechanisms are capable of clustering all the telomeres, in
no case could a bouquet self-organize in a fashion resembling the abrupt transition seen
in time courses without an additional symmetry-breaking condition. At least one of the
following additions were required: initial polarization of centromeres and telomeres, a
force acting to pull telomeres to one location, or a gradient of attraction between
chromosome regions increasing towards the telomeres. We are currently fine-tuning the
48
parameters of the bouquet-forming models in an attempt to replicate the time course data
more accurately.
C2.80 - Reorganization and polarization of the meiotic bouquet-stage cell can be
uncoupled from telomere clustering
Carrie Cowan(1) and W. Zacheus Cande(1,2)
(1) Dept. of Plant & Microbial Biology University of California - Berkeley (2) Dept. of Molecular Cell
Biology University of California - Berkeley
The bouquet stage of meiotic prophase involves striking cellular and nuclear
rearrangements. We have developed a method of culturi ng plant reproductive organs in
vitro in order to address meiotic reorganization, specifically the changes in telomere and
nuclear pore distributions and cellular architecture. Telomeres cluster to a single site on
the nuclear envelope during meiotic prophase, the classic definition of the bouquet stage.
The majority of cytoplasmic microtubules were found to be focused opposite the
telomere cluster during the bouquet stage. Additional asymmetry was apparent in the
eccentric placement of the nucleus and the orientation of the telomere cluster toward the
cell cortex. We found that nuclear pores reorganized from a uniform distribution around
the entire nuclear periphery into a single group opposite the clustered telomeres at the
bouquet stage. The positions of the nuclear pores relative to the telomeres and to the cell
were found to be constrained, demonstrating nuclear polarization. Treatment with
colchicine inhibited telomere clustering; telomeres were found at the nuclear periphery,
distributed randomly in one hemisphere of the nuclear surface. Nuclear pore clustering
was not affected by colchicine treatment, demonstrating that nuclear pores can cluster
independently of telomeres. Nuclear pores were positioned normally with respect to the
cell cortex, despite the failure of telomere clustering, indicating that a polaritydetermining mechanism exists independent of telomere position. This work presents
novel evidence of meiotic polarization and suggests that telomeres may respond to an
asymmetry already present in the cell at the time of bouquet formation.
C2.81 - Motility of paired chromosomes during meiotic prophase
Carrie Cowan(1), Pete Carlton(2), Eric White(3), David Kaback(3), and W. Zacheus
Cande(1,2)
(1) Dept. of Plant & Microbial Biology University of California - Berkeley (2) Dept. of Molecular Cell
Biology University of California - Berkeley (3) Dept. of Microbiology & Molecular Genetics UMDNJ
The extent of chromosome movement within the non-dividing nucleus is beginning to be
untangled. Chromosomes in the interphase nuclei of Saccharomyces cerevisiae and
Drosophila melanogaster have been shown to exhibit only thermal (Brownian) motion by
49
investigating the behavior of chromosomal loci tagged with GFP fusion constructs. The
meiotic prophase nucleus presents an interesting arena for further investigations of
chromosome movement. Meiotic prophase performs numerous specialized tasks, such as
chromosome synapsis and recombination, which may involve specialized chromosome
movements. We chose to investigate synapsed chromosomes in the Saccharomyces
cerevisiae meiotic nucleus. Using a GFP fusion to a synaptonemal complex component
(zip1-GFP), time-lapse video microscopy, and three-dimensional modeling, we have
found that S. cerevisiae meiotic chromosomes undergo extensive motility within the
nucleus. Analysis of zip1-GFP revealed random walks, sustained trajectories, and
flexing. We present an analysis of these novel chromosome movements and discuss their
drug sensitivities, allowing speculation as to their origin.
C2.82 - M31 and macroH2A1.2 form a macromolecular chromatin complex at the
pseudoautosomal region (PAR) during mouse meiosis
J. M. A. Turner and P. S. Burgoyne, National Institute for Medical Research, London, UK; P. B.
Singh, Roslin Institute, Edinburgh, UK
During male meiosis in a number of organisms, the sex chromosomes become
transcriptionally inactivated and form a structure called the sex- or XY body. It has been
proposed that inactivation of the X chromosome during male meiosis is mechanistically
related to somatic X inactivation in females and that inactivation of the Y occurs by
spreading of heterochromatinisation from the X to the Y chromosome via the
pseudoautosomal region (1,2). Here we have investigated this ‘trans-heterochromatinisation’ model in more detail, using meiotic preparations from normal and sex
chromosomally variant mice, immunostained for the heterochromatin-associated proteins
M31 and histone macroH2A1.2. In addition to the previously reported localisation of
these proteins to the sex body, both proteins also localised to a focus within the region of
distal PAR that includes the steroid sulfatase gene, suggesting that the PAR assembles a
discrete macromolecular chromatin complex. While this focus satisfies one prediction of
the trans-heterochromatinisation model, further analysis showed that this focus is present
whether or not there is sex chromosome inactivation. Other studies have shown that
distally located autosomal chiasmata are associated with increased non-disjunction. Since
the X-Y bivalent always has an extremely distal chiasma, we suggest that this
macromolecular chromatin complex may play a role in maintaining X-Y association and
thus normal X-Y disjunction.
3. Ayoub et al. (1997). Chromosoma 106, 1-10.
4. Motzkus et al. (1999). Cytogenet. Cell Genet. 86, 83-88.
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C2.84 - Massive polycomplexes in ndt80 mutants are probably functional
synaptonemal complexes
H. Bhuiyan and K. Schmekel, Dept. Molecular Biology and Functional Genomics, Stockholm
University, SE-106 91 Stockholm, Sweden
Structures that morphologically resemble stacks of synaptonemal complex (SCs),
polycomplexes (PCs), may occur before or after pachytene in normal cells, but are more
common in meiotic mutants. PCs have been believed to be complexes of chromosome
dissociated SCs.
We have investigated the ndt80 mutant in yeast, which arrest in pachytene. The
SCs in ndt80 appear structurally similar to those of wild type in preparations of spread
chromosome. However, sections of in situ embedded intact cells show in electron
microscope that essentially all SCs are organized in massive PCs. In a single section, PCs
may contain up to nine individual SCs, forming unilayer sheets. Serial sectioning of
ndt80 nuclei show that every nucleus that contain SCs have the SCs aggregated as PCs,
very few SCs are disconnected from PCs and only short pieces of SCs are protruding out
from the PC. On an average PC occupies about half the diameter of an nucleus. Random
scoring of nuclei in single sections shows that about 75% of the nuclei contain PCs. A
FISH experiment using a chromosome specific probe (end of chr. III) shows pairing of
homologous chromosomes (75%). Preliminary results of immuno-EM-labeling show that
DNA may be present inside the PCs. In conclusion, the SCs in these PCs seem to be
involved in chromosome synapsis. Spreading of chromosomes obviously disrupts nuclear
organization.
C2.85.- Multicolour painting of mouse meiotic chromosomes
Henry H.Q. Heng1, Guo Liu1, Steve Bremer1, Christine J. Ye2, Mark Hughes1 and Peter
Moens3
1
Centre for Molecular Medicine and Genetics, Dept. Pathology, Karmanos Cancer Inst., Wayne State
University, Detroit, Michigan, USA, 2 SeeDNA Biotech Inc, Windsor, Ontario, Canada, 3 Dept. Biology,
York University, Toronto, Ontario, Canada
Spectral karyotyping (SKY) uses chromosome-specific probes for in situ hybridization to
colour code each of the 21 mouse or 24 human chromosomes. While it has been used
extensively in the recognition of mitotic chromosomal aberrations, we have now adapted
SKY chromosome painting to identify meiotic chromosomes during pachytene and
metaphase I. We expect the technique will facilitate the detection of non-homologous
associations at meiotic prophase as well as chromosome-specific structural and
behavioural characteristics.