Chromosoma (2002) 111:96–105
DOI 10.1007/s00412-002-0193-5
O R I G I N A L A RT I C L E
Susan George · Pearl Behl · Rhoda deGuzman
Marian Lee · Stefan Rusyniak · Yasuo Hotta
Kazuyuki Hiratsuka · Hisabumi Takase
Clare Hasenkampf
Dmc1 fluorescent foci in prophase I nuclei of diploid,
triploid and hybrid lilies
Received: 7 November 2001 / Revised: 7 March 2002 / Accepted: 11 March 2002 / Published online: 15 May 2002
© Springer-Verlag 2002
Abstract We examined the distribution of meiotic
epitopes for the Dmc1 protein of lilies in a normal diploid, a triploid, and in a diploid species-hybrid. The triploid has an extra chromosome set; all three sets align, but
only two of the three axes intimately pair at a given location. Our findings with the triploid support the idea that
retention of the foci until the pachytene stage requires a
successful homology check and synaptonemal complex
(SC) initiation; the number of foci in the triploid diminishes by approximately 30% from early zygotene to
pachytene, and the triploid pachytene values are similar
to the pachytene values of the diploid. The specieshybrid lacks chromosome homology, has reduced SC
formation and few reciprocal genetic exchanges. In this
species-hybrid the number of foci at early zygotene is
similar to that in the normal diploid but is dramatically
reduced by mid-zygotene. The extent to which the number of Dmc1 foci is reduced is similar to the extent that
SC formation is reduced. In contrast the extent of the
reduction in reciprocal genetic exchange in the specieshybrid is much greater than the reduction in the number
of foci. We conclude that Dmc1 protein is involved in
Edited by: P. Moens
We would like to dedicate this article to the memory of Dr. Herbert
Stern. He provided us (Y. Hotta and C. Hasenkampf) with an exemplary role model – both as a rigorous, successful scientist and as a
kind and witty person of the highest integrity. We miss him dearly.
S. George · P. Behl · R. deGuzman · M. Lee · S. Rusyniak
C. Hasenkampf (✉)
Division of Life Science, University of Toronto at Scarborough,
1265 Military Trail, Scarborough, Ontario M1C 1A4, Canada
e-mail: [email protected]
K. Hiratsuka · H. Takase
Department of Molecular Biology,
Graduate School of Biological Sciences,
Nara Institute of Science and Technology,
8916-5 Takayama, Ikoma, Nara 630-0101, Japan
Y. Hotta
Department of Nutrition and Health,
Niigata University of Health and Welfare,
1398 Shimami, Niigata, Niigata 950-3198, Japan
homology checking, but the impact of failure to find
homology affects SC formation and reciprocal genetic
exchange differentially.
Introduction
Meiosis is a specialized type of nuclear division during
which the chromosome number is reduced in anticipation of fertilization. The reduction in chromosome number is accomplished by having one round of chromosome
replication be followed by two rounds of chromosome
division. In the first meiotic division homologous chromosomes are separated; in the second meiotic division
sister chromatid centromeres are separated. Ensuring that
homologous chromosomes are separated requires that
specialized events occur in both premeiotic S-phase and
in the first meiotic prophase.
In a standard meiotic prophase I homologous chromosomes come together, pair up precisely along their axial
length, and form an elaborate tripartite structure known
as the synaptonemal complex (SC). While homologous
SC formation is the rule, an SC can form between nonhomologous chromosome segments in a variety of
circumstances [e.g. species-hybrids (Toledo et al. 1979),
synaptic accommodation (Moses and Poorman 1981),
haploids (Loidl et al. 1991), meiotic mutants (Yoshida
et al. 1998)].
A pathway for repairing double-strand breaks is also
present during prophase I. Double-strand breaks in the
DNA are created, then processed to expose re-sected single-stranded DNA. The 3′ ends of such single strands then
become associated with RecA-like proteins. D-loop formation proceeds between the single-stranded DNA (associated with RecA-like proteins) and the double-stranded
DNA of the homologous chromosome, and a double
Holliday junction can be formed. Resolution of the Holliday junction in a particular manner can result in reciprocal
genetic exchange (reviewed in Davis and Smith 2001).
One RecA-like protein found in eukaryotes is the
Rad51 protein. In vitro Rad51 can form a nucleoprotein
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filament with single-stranded DNA (Ogawa et al. 1993).
Another RecA-like protein, Dmc1, has been found in
many eukaryotes. In contrast to RecA protein and other
RecA-like proteins, conditions under which Dmc1 forms
a nucleoprotein filament with single-stranded DNA in
vitro have not yet been found. Instead, in vitro, Dmc1
forms octameric rings or stacks of rings (Masson et al.
1999; Passy et al. 1999). Both Rad51 and Dmc1 can
bind, in vitro, to single-stranded DNA but neither protein
does so as robustly as RecA protein, and it is likely that
accessory proteins would be needed for Rad51-mediated
strand invasion in vivo (Gupta et al. 1999, 2001).
Proper functioning of Rad51 protein is important to
homologous recombination both in a mitotic cycle (for
repair of DNA damage) and during meiosis. Use of antibodies directed against Rad51 protein has revealed that
distinct foci of Rad51 form in response to DNA damage
during a mitotic cycle (Gasior et al. 2001) and during
meiotic prophase I (Bishop 1994). In yeast, null mutants
of Rad51 have increased sensitivity to radiation, and
have reduced levels of recombination during meiosis
(Shinohara et al. 1992). In mice, loss of Rad51 function
results in embryonic lethality (Lim and Hasty 1996).
Dmc1 generally appears to be meiosis specific (Masson
and West 2001). In Dmc1 knockout mice both male and female sterility is observed (Yoshida et al. 1998). In Saccharomyces cerevisiae, Dmc1 null mutants have a reduction in
recombination (Bishop et al. 1992). Use of anti-Dmc1 antibodies reveals that Dmc1 protein, like Rad51 protein, occurs in distinct foci during prophase I (Bishop 1994). The
relationship of Rad51 foci and Dmc1 foci has been most
extensively studied in S. cerevisiae and mammals. Bishop
(1994) found that in an SK1 strain of S. cerevisiae, Dmc1
and Rad51 foci co-localize in wild-type individuals, but
foci for either can form in the absence of the other [i.e.
Rad51 foci can form in yeast Dmc1 null mutants (Bishop
1994); Dmc1 foci can form in Rad51 null mutants, although the foci are less bright than in wild type (Shinohara
et al. 2000)].
Analysis of mutants suggests that Rad51 protein and
Dmc1 protein are also important in stabilizing the association of homologous chromosomes prior to the formation
of SCs. Rockmill et al. (1995) have suggested that Rad51
and Dmc1 are involved in a chromosome homology
check. If homology is present, then somehow a stable axial association can form between homologous chromosomes. They further propose that this axial association
expedites subsequent SC formation. In fact Rad51 or
Dmc1 null mutants do have delayed and reduced amounts
of SC. Further it is likely that some of the SC that forms
is nonhomologous. Rad51 knockout mice die, so their
meiotic phenotype cannot be assessed. Knockout Dmc1
mice are sterile. Cytological analysis of these mice reveals that the homologous chromosomes generally fail to
form SC; of the small amount of SC that is present, some
is likely nonhomologous (Yoshida et al. 1998). Anderson
et al. (1997) (in lily) and Tarsounas et al. (1999) (in mice)
have found Rad51 and Dmc1 to be associated with components of the SC both before and after mature SC has
formed, and postulate that these proteins aid in the formation of axial associations. Thus in addition to their roles
in the double-strand break pathway, Dmc1 and Rad51
proteins appear to be important for the formation of axial
associations between homologous chromosomes, and in
expediting timely SC formation.
Strategies for studying Dmc1 function to date have
been largely 'trans' type analyses. The function and distribution of both wild-type and mutant Dmc1 protein have
been examined. We elected to use an alternate approach.
We sought to elucidate further the function of the wildtype Dmc1 protein in normal lily diploid meiosis and
compare it with events in lilies with abnormal chromosome situations. We decided to study the distribution
of Dmc1 epitopes in a triploid lily with an extra set of
chromosomes, and in a diploid species-hybrid known as
Black Beauty. Black Beauty diploid hybrids have a very
reduced level of chiasma formation (Emsweller and
Uhring 1966). The problems that arise during meiosis in
this diploid hybrid are likely due to lack of chromosome
homology, and not genic imbalances between the two parental genotypes. This is inferred from the fact that if the
chromosome number of the diploid hybrid is doubled, the
synthetic allotetraploid undergoes a normal meiosis with
a high degree of reciprocal genetic exchange (Toledo
et al. 1979). Here we report differences in the number of
Dmc1 foci seen in our control diploid, in the triploid, and
in the species-hybrid. We discuss these findings relative
to the function of the Dmc1 protein.
Materials and methods
The lilies used in this study were either Lilium tigrinum (diploid),
L. tigrinum (triploid), or the cultivar Black Beauty, which is a species-hybrid that results from a cross of Lilium henryi with Lilium
speciosum. The Tigrinum lilies were purchased from Van Hof and
Blokker bulb distributors (Mississisauga, Ontario), and the Black
Beauty bulbs were purchased from Dahlstrom and Watt Bulb
Farms (Smith River, Calif.). All plants were grown in a greenhouse under conditions of natural lighting, with a day/night temperature regimen of 25°C/15°C.
Squash preparations for examining chromosome morphology
were prepared by squeezing the contents of individual buds into a
fixative consisting of three parts 95% ethanol, and 1 part glacial
acetic acid for 30 to 120 min. The meiotic cells came out of the
anther as a packed filament of connected cells. Portions of the filament were removed from the fixative and placed on a clean slide
with iron aceto-carmine. The material was stained for 10 min as it
was uniformly distributed on the slide. A cover glass was then
added, and pressure was applied to produce the squash preparation. The slides were dipped in liquid nitrogen, and the cover glass
was removed. Preparations were then dehydrated in a graded ethanol series consisting of 70% ethanol, two changes in 95% ethanol,
and two changes of 100% ethanol. Slides were placed in histoclear
(DiaMed) for 3 min, then drained and mounted with a drop of Permount and a cover glass. Nuclei in prophase I and metaphase I
were examined using a Zeiss axiophot microscope equipped with
a 63× planapo lens. The criteria for determining the substages of
prophase I were a combination of the criteria of Erickson (1948)
and Holm (1977), as indicated in Hasenkampf et al. (1992). The
triploid and species-hybrid each had the potential for incomplete
synapsis. Therefore the shape and location of the nucleoli, and the
overall extent of chromosome condensation were particularly important criteria.
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For the immunocytochemical analysis floral buds at leptoteneearly zygotene, mid-late zygotene, and early pachytene were used.
One anther of a bud was used to prepare a squash preparation to
determine the meiotic stage. The remainder of the anthers were
used for immunocytochemistry if the bud was at the desired stage.
For the immunocytochemistry the meiotic nuclei were prepared in
a manner similar to the procedure used by Terasawa et al. (1995).
The contents of the anthers were squeezed into a small tube containing 4% paraformaldehyde dissolved in phosphate-buffered
saline (PBS) at pH 7.4 (PBS is 130 mM NaCl, 7 mM Na2HPO4,
3 mM NaH2PO4). After fixation for 10 min at room temperature,
the fixative was removed and the filaments were washed three
times in a solution consisting of 0.6 M mannitol, 5 mM 2-Nmorpholino ethane sulfonic acid, 50 mM EDTA, 0.15 mM spermine, pH 5.6. The filaments of cells were then incubated in the
above solution to which Y-23 pectolyase (Sigma) had been added
to a final concentration of 2%. After 5 min the solution was replaced with 4% paraformaldehyde in PBS and the cells were fixed
again for 30 min. Next a small portion of a filament in 25 µm
of fixative was placed on a clean slide, and 25 µm of 0.4% Triton
X-100 was added. After 2 min a cover glass was added and the filament was gently tapped into a monolayer to disperse the meiotic
cells evenly, and pressure was applied to the cover glass to squash
open the cells. Slides were then dipped in liquid nitrogen, and the
cover glasses were removed. Slides were immediately transferred
to a tray containing PBS and were washed three times, each wash
lasting 3 min. For immunocytochemistry, individual slides were
removed from the tray, the PBS was blotted away, and 200 µm of
blocking solution was applied. The blocking solution consisted of
0.5% blocking reagent (Roche), and 5% goat serum, both dissolved in double-distilled water. After 30 min, the blocking solution was shaken off the slide, and 200 µm of incubation buffer
(0.5% blocking solution with 1% goat serum) containing the primary antibody was added to the slide; slides were incubated for
2 h at room temperature.
Three different primary immune sera were used. For the positive controls the primary antibody solution was an immune serum
designated 411, directed against lily histone H1 (Riggs 1994). The
negative control experiments were done with the preimmune serum from the rabbit later used to generate immune serum 1. Two
different immune sera were used to detect Dmc1 epitopes. The
first immune serum was prepared using the entire lily Dmc1 protein. Immune serum 1 reacts with purified lily Dmc1 protein, and
with the purified Dmc1 protein of Arabidopsis. It does not react
with purified Arabidopsis Rad51 protein. Immune serum 1 reacts
with only one band in our protein gels; however, we do not have
purified lily Rad51 protein and cannot absolutely eliminate the
possibility that immune serum 1 reacts with both lily Dmc1 and
Rad51 proteins. The other Dmc1 immune serum was kindly provided by the laboratory of Dr. Tomoko Ogawa. The Ogawa immune serum was created using a synthetic peptide corresponding
to the 18 amino acids at the N-terminus of the lily Dmc1 protein
(the lily gene was originally called LIM15; Kobayashi et al.
1993). This immune serum is specific for the Dmc1 protein and
does not react with lily Rad51 protein (Terasawa et al. 1995;
Anderson et al. 1997). All immune sera were diluted 1:750 for the
immunocytochemistry experiments.
Once the incubation in primary antibody was completed, slides
were washed four times (5 min per wash) in PBS with 0.1%
Tween-20, followed by two washes in PBS, then one wash in incubation buffer. Slides were then drained briefly and 200 µm of
secondary antibody was added. The secondary antibody was a
goat, anti-rabbit IgG conjugated to fluorescein (Vector Laboratories) diluted 1:100 in incubation buffer. After a 2 h incubation at
room temperature the slides were washed in PBS four times, each
wash lasting 3 min. The washing buffer was dabbed off the slides
and a drop of aqueous Vectashield mounting media (Vector Laboratories) was added. Some slides were stained with 4′,6-diamidino-2-phenylindole (DAPI) before mounting. Slides were observed using a 100× Zeiss planapochromat lens. The fluorescein
signal was detected with the fluorescein filter set (Zeiss 487909);
the DAPI signal was detected using Zeiss filter set 487902. Re-
sults from 20 nuclei were used for each stage for immune serum 1,
and results from 10 nuclei for each stage were used for the immune serum 2 (Ogawa).
For the quantification of foci the fluorescein filter set was
used, and the nuclei were positioned in the center of the field of
view. The primary focal plane was selected. The focused image
was captured using a Sony 3 CCD color video camera. The resulting image was digitized and analyzed using Northern Eclipse Software (Empix, Missassauga, Ontario). The boundary of the nucleus
in the digitized image was delimited on the image and the number
of discrete fluorescent foci within the delimited region was determined. The color images were then converted to black and white
for publication. Since the analyzed nuclei had been liberated from
the cell and had been squashed into a flat preparation, most foci
were visible within the selected focal plane. However some outlying foci may not have been in the captured focal plane. Hence our
estimates of the number of foci are minimum estimates.
Immunoblots were prepared from samples of approximately
five to ten buds per stage; each bud contained thousands of meiotic cells. One anther per bud was used for cytological assessment
of the meiotic stage. The remaining anthers of each bud of the
appropriate stage, were used for immunoblotting. The contents of
each anther were squeezed into ice-cold White's plant tissue culture medium (White 1963), pH 5.4, supplemented with protease
inhibitors according to the manufacturer's instructions (Roche
catalog no. 1873580). Once a collection for a particular sample
was complete, the White's medium was removed, and the meiotic
cells were homogenized in an equal volume of 2×DB (2% SDS,
0.6% w/v β-mercaptoethanol, 20 mM TRIS, pH 8.6, 20% glycerol). The protein concentration of each sample was determined.
Gel electrophoresis and immunoblotting were done as indicated in
Hasenkampf et al. (1992).
To confirm the protein concentration estimates and assess the
quality of the preparations, 5 µg of each protein sample were subjected to SDS-polyacrylamide gel electrophoresis and stained with
Coomassie Brilliant Blue. If the concentration of the core histones
was the same in each protein extract, then additional 5 µg samples
were used in gels to produce the immunoblots. While no direct
loading controls were present for the individual lanes of each gel
used to produce the immunoblots, the protein concentrations of
each sample were confirmed (as indicated above), and the immunoblot series done for each type of lily was repeated at least three
times for immune serum 1, and at least twice for immune serum 2
(Ogawa). For each type of lily the replicates of the immunoblots
showed the same trend.
Results
Bright-field microscopy
A light microscopy study was performed to access
the extent of pairing and reciprocal genetic exchange in
the normal diploid tiger lily, in the triploid tiger lily, and
in the species-hybrid Black Beauty. All three types of
lilies had a high degree of pairing during pachytene
(Fig. 1A, C, E). Metaphase I analysis was done for each
type of lily to determine the extent of reciprocal genetic
exchange. For the metaphase I analysis 30 nuclei (from
three different plants) were examined for each type of
plant. For each nucleus the numbers of univalents, bivalents, and trivalents were recorded.
The diploid tiger lily served as the positive control,
being expected to have normal homologous chromosome
pairing, synapsis, and reciprocal genetic exchange. In the
normal diploid, two by two alignment was followed by
more intimate pairing such that each pair of chromo-
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Fig. 1A–F Pachytene and
metaphase I chromosome configurations. A, B Micrographs
of pachytene and metaphase I
nuclei of the diploid control.
C, D Micrographs of the triploid, Lilium tigrinum. The
arrowheads in C point to
regions where all three homologs are aligned and have axial
associations. In D, 11 trivalents, 1 bivalent (II), and one
univalent (I) can be seen.
E, F Micrographs of the species-hybrid. The nucleus in F
has 16 univalents, and 4 bivalents (II). Bar in E represents
10 µm for A, C, and E; bar in
F represents 20 µm for B, D, F
somes appeared as a single uninterrupted bivalent. As
expected, the normal diploid formed 12 bivalents (e.g.
Fig. 1B) in all nuclei examined, indicating successful
completion of all events necessary to produce reciprocal
genetic exchange. While a precise count of the number
of reciprocal exchanges was not undertaken, two or more
exchanges per bivalent were seen in all 30 cells examined, and the chiasma frequency is likely very similar to
the reported value of 54.8±6.0 per nucleus (Stack et al.
1989).
In the triploid all three homologs regularly aligned
three by three, but the more intimate synapsis occurred
two by two. Thus each set of (three) aligned chromosomes had chromosome regions where one chromosome
axes was not synapsed (Fig. 1C, arrowheads). This result
is similar to that of Loidl and Jones (1986) in an Allium
autotriploid, and that of Moens (1968) in a triploid tiger
lily. The lily triploid is capable of forming a wide variety
of types of chromosome configuration at metaphase I,
depending on the number of reciprocal genetic exchanges, and which two (of the three) chromosomes participate in each exchange. The most common situation
found was 11 trivalents, 1 bivalent, and 1 univalent
(Fig. 1D). This configuration was seen in 16 of the 30
nuclei examined. In 11 of the 30 nuclei all 12 sets of
three homologs were associated as 12 trivalents. The remaining 3 nuclei had 10 trivalents, 2 bivalents, and 2
univalents. Thus in general there was a high degree of
trivalent formation, indicating that there was a high degree of reciprocal genetic exchange, and that most often
all paired chromosome arms participated in a genetic exchange. Therefore the presence of an extra set of chromosomes, and the occurrence of unsaturated SC formation (one-third of the total axial length was aligned but
not synapsed) did not block the ability of the nuclei to
accomplish high levels of reciprocal genetic exchange.
Black Beauty is a diploid species-hybrid with 24
chromosomes, 12 each from two different but related
species. Thus the chromosomes are homeologous, not
strictly homologous. The homeologous chromosomes
accomplish pairing to a high degree (Fig. 1E); unpaired
regions were only occasionally seen. Previous ultrastructural analyses of diploid Black Beauty indicated that it
forms an SC (Toledo et al. 1979, and Hasenkampf and
Shull, unpublished results). Even though the appearance
of the SC is normal, the extent of SC formation in
diploid Black Beauty is reduced. Toledo et al. (1979)
estimate that SC formation is 64% complete in diploid
Black Beauty, and Hasenkampf and Shull (unpublished
results) estimate that SC formation only includes 37%
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Fig. 2A, B Immunoblot profiles in diploid, triploid and specieshybrid lilies. Five micrograms of nuclear proteins were loaded in
each lane. The blots in A were probed with immune serum 1. This
immune serum was raised against the entire Dmc1 protein. The
blots in B were probed with immune serum 2 (Ogawa), which is
specific for Dmc1 protein. A, B Lane 1 premeiotic S-phase, lane 2
leptotene-early zygotene, lane 3 mid- to late zygotene, lane 4
pachytene (early, mid- and late), lane 5 post-pachytene stages
ranging from diakinesis through the second meiotic division. The
top molecular weight marker (lane M) represents 32.5 kDa, and
the lower marker represents 25 kDa
of the paired chromosome length. Previous reports indicated that Black Beauty diploids are largely achiasmatic
(Emsweller and Uhring 1966; Toledo et al. 1979). We
found a similar result in our study. In the metaphase I
nuclei examined most chromosomes occurred as univalents. Occasional bivalents were observed (Fig. 1F, bivalents indicated by roman numeral II). Three nuclei had
no bivalents; eight nuclei had two bivalents; eight nuclei
had three bivalents; five nuclei had four bivalents; five
nuclei had five bivalents; and one nucleus had six. Thus
of the 12 potential bivalents that could form if reciprocal
genetic exchange occurred normally, most nuclei had only two or three bivalents.
In summary, the diploid control exhibited normal
pairing and reciprocal genetic exchange. The triploid lily
had three by three alignment of the chromosomes, with
pairing partner switches, and a high degree of reciprocal
genetic exchange. Trivalent formation was the norm at
metaphase I indicating that any two of the three homologs could participate in the exchange. The diploid species-hybrid had largely normal chromosome pairing, but
greatly reduced levels of reciprocal genetic exchange.
Immunoblots
Protein extracts for immunoblotting were prepared from
lily anthers at a variety of premeiotic and meiotic stages
for each of the three lily varieties. The stages examined
were the premeiotic interphase, leptotene-early zygotene,
mid-zygotene, pachytene (early, mid- and late) and diplotene-diakinesis. Results of immunoblotting are shown
in Fig. 2A, B. Immune serum 1 and immune serum 2
gave similar results, except that immune serum 1 regularly gave the stronger signal.
For the diploid control and the triploid lily the results
were similar. That is, the strongest immunostaining was
observed in the leptotene-early zygotene samples, it declined in mid-zygotene and was gone (or reduced) in the
pachytene extracts. Results obtained with the specieshybrid Black Beauty were different. Immunostaining
was only detectable in the leptotene-early zygotene samples. Immune serum 1 had a modest amount of staining
at this early stage, but immune serum 2 had only a barely
perceptible band. This was the first indication that the
Dmc1 protein might be behaving differentially in an unusual chromosomal setting; further differences were seen
in the immunocytochemistry results.
Immunocytochemistry
Fluorescent immunocytochemistry was undertaken using
squash preparations of chromosomes at early zygotene,
mid-zygotene and early pachytene. Positive control
slides were treated with anti-histone H1 immune serum
to ensure that squash preparations for all three lily types,
at all stages, were immunoreactive. As expected, uniform staining of the chromatin was observed for all three
varieties at these three stages (data not shown).
Immunostaining was seen for all three lily varieties,
at all three stages examined, with both Dmc1 immune
sera (Immune serum 1, Fig.3, Immune serum 2, Fig.4).
With each immune serum, two patterns of staining were
seen. There was a uniform diffuse staining of the chromatin (also seen in Franklin et al. 1999). This diffuse
staining was not seen in the negative control slides treated with preimmune serum and thus does not represent
background staining. In addition to the diffuse staining,
many bright discrete foci were seen along the axes of the
chromosomes similar to foci reported in other immunocytochemical studies of Dmc1 and Rad51 proteins (e.g.
Bishop et al. 1992; Bishop 1994; Terasawa et al. 1995;
Dresser et al. 1997; Tarsounas et al. 1999). To confirm
the position of the foci on the chromosomes, preparations were stained with DAPI and the images were compared using DAPI and fluorescein filters (data not
101
shown). Corresponding images show that the foci are
along the chromosomes as observed in numerous other
studies (e.g. Terasawa et al. 1995; Yoshida et al. 1998;
Tarsounas et al. 1999).
Foci in the normal diploid lily
Fig. 3A–I Foci in lily nuclei reacted with immune serum 1.
A, D, G Micrographs of the diploid control, L. tigrinum. B, E, H
Micrographs of the triploid, L. tigrinum. C, F, I Micrographs of
the species-hybrid, Black Beauty. A, B, C Early zygotene nuclei.
D, E, F Mid-zygotene nuclei. G, H, I Early pachytene. Bar represents 10 µm for all panels
Fig. 4A–I Foci in lily nuclei reacted with immune serum 2.
A, D, G Micrographs of the diploid control, L. tigrinum. B, E, H
Micrographs of the triploid, L. tigrinum. C, F, I Micrographs of
the species-hybrid, Black Beauty. A, B, C Early zygotene nuclei.
D, E, F Mid-zygotene nuclei. G, H, I Early pachytene. Bar represents 10 µm for all panels
In addition to the qualitative assessment, we also quantified the number of foci seen per nucleus for both immune
sera. To assess our ability to detect foci we compared the
estimate we obtained with our diploid control, L. tigrinum, with that obtained by Terasawa et al. (1995), using
diploid Lilium longiflorum. They report the values for
Dmc1 foci at zygotene as being between 800 and 1000.
Using the same Dmc1 immune serum(our immune serum
2) we obtained a mean value of 656 foci (SD = 51). Our
somewhat lower zygotene value may be due to speciesspecific differences between L. longiflorum and L. tigrinum, or may be due to a failure, on our part, to detect foci
outside the main focal plane. Using our other immune serum on the early zygotene nuclei of our diploid control,
we observed a mean value of 1015 foci per nucleus
(SD = 162).
We compared the number of Dmc1 foci seen at early
zygotene, mid-zygotene and pachytene in our diploid
plants using the two immune serum. The results of our
comparisons are illustrated in Fig. 5A. The bar graphs
represent the mean number of foci observed for the diploid at each of the three stages examined, for each immune serum. As can be seen in Fig. 5A, the two immune
serum generally revealed the same trend – a reduction in
number of foci from early zygotene to early pachytene.
However, there was one notable difference between the
two sera. Immune serum 1 always detected more foci
than did immune serum 2. Immune serum 2 was raised
against the 18 amino acids at the N-terminus of the lily
Dmc1 protein. Immune serum 1 was raised against the
entire Dmc1 protein. Thus immune serum 1 likely is detecting a greater range of Dmc1 epitopes. Hence immune
serum 1 is likely the more sensitive immune serum,
capable of recognizing less bright collections of Dmc1
proteins. The difference in number of foci detected with
the two sera was most pronounced in the early zygotene
nuclei, then decreased such that by early pachytene the
number of foci detected with both serum was very similar. We interpret this to mean that early in the process of
pairing (i.e. in early zygotene) there is a set of bright foci
detectable with both immune sera, and a set of less
bright foci that are only detectable with immune serum
1. Then as the pairing process progresses, the less intense foci either disappear or merge.
Foci in the diploid control compared with foci
in the triploid, and in the species-hybrid
The triploid lily (Fig. 5B), like the diploid, shows a reduction in number of foci from early zygotene to early
102
Fig. 5 Distribution of foci at different prophase I stages. The
mean numbers of foci for each stage, for each type of lily sample,
are presented as a series of bar graphs. Solid bars values obtained
using immune serum 1; hatched bars values obtained with immune serum 2; eZ early zygotene, mZ mid-zygotene, P early
pachytene. A Diploid L. tigrinum. For the nuclei reacted with immune serum 1 the standard deviations for the eZ, mZ and P samples were 162, 161, and 79, respectively. Paired t-tests were done,
and the observed reductions in mean number of foci from eZ to
mZ, and mZ to P were statistically significant (P<0.005 and
P<0.001, respectively). For immune serum 2 the standard deviations for the eZ, mZ and P samples were 51, 52 and 119, respectively. The observed, small differences in the stage-specific mean
values were not statistically significant. B Triploid L. tigrinum.
For the nuclei reacted with immune serum 1 the standard deviations for the eZ, mZ and P samples were 79, 102 and 166, respectively. All of these stage-specific differences in foci number were
statistically significant (P<0.001). For immune serum 2 the standard deviations for the eZ, mZ and P samples were 109, 113 and
78, respectively. The difference in number of foci from early zygotene to mid-zygotene was not statistically significant; the reduction from mid-zygotene to early pachytene was (P<0.001). C Diploid species-hybrid, Black Beauty. For the nuclei reacted with immune serum 1 the standard deviations for the eZ, mZ and P samples were 127, 96, and 71, respectively. All of the stage-specific
differences in mean number of foci were statistically significant
(P<0.001). For immune serum 2 the standard deviations for the
eZ, mZ and P samples were 105, 42, and 52, respectively. The reduction in number of foci from eZ to mZ was statistically significant (P<0.001), whereas the small reduction from mid-zygotene to
early pachytene was not
pachytene. The triploid has 33% more chromosome axis
than the diploid, and at early and mid-zygotene has
somewhat more foci than the diploid. Nonetheless by
early pachytene the triploid has approximately the same
number of foci as the diploid. Thus the number of bright
foci detectable at early pachytene is related to the synapsed chromosome length, rather than the total chromosome length present.
Lastly we examined the foci pattern in the diploid
species-hybrid Black Beauty (Fig. 5C). At early zygotene the diploid species-hybrid has approximately 80%
as many foci as the normal diploid at the same stage.
However, in mid-zygotene there is a dramatic reduction
in foci number, such that by early pachytene the specieshybrid has only 43% as many foci as the diploid control.
As can be seen in Fig. 3F, I and Fig. 4F, I, the Black
Beauty nuclei at mid-zygotene and early pachytene have
a smaller volume than either the diploid or triploid nuclei
at the same stage. It is unclear why the paraformaldehyde-fixed Black Beauty nuclei at these stages resisted
squashing, but this phenomenon was seen for most of the
Black Beauty nuclei examined. Nonetheless we do not
think the smaller nuclear volume significantly affected
our ability to count the foci; we think the observed reductions were real. This view is supported by the Black
Beauty immunoblot results (Fig. 2, right-most blots).
The Dmc1 protein is only detectable in the leptoteneearly zygotene protein extracts of the Black Beauty species-hybrid and was difficult to detect at all with immune
serum 2. Thus we conclude that the species-hybrid is unable to retain a normal level of Dmc1 foci beyond early
zygotene.
Discussion
We have examined three different varieties of lilies with
three different meiotic 'conditions'. We first examined
the temporal distribution of Dmc1 epitopes in our control diploid using immunoblotting. We found the highest
level of immunostaining in the leptotene-early zygotene
extracts. Staining declined during zygotene, and was
usually absent in the pachytene extracts. Our immunoblot results are similar to those obtained by Terasawa
et al. (1995) except that they observed a low level of
staining in their pachytene extracts. This difference may
be due to the fact that our pachytene extracts are a mixture of early, mid- and late pachytene, and their extract
may have been of only early pachytene. This interpretation is consistent with the results from our immunocytochemistry. We selected very early pachytene nuclei for
the immunocytochemical analysis and we did observe
immunostaining in the early pachytene nuclei.
Dmc1 foci and synapsis
The triploid tiger lily is able to undergo chromosome
alignment, intimate pairing and reciprocal genetic exchange; its only observed cytological difference from the
control was that approximately one-third of its axial
length was excluded from intimate association and reciprocal genetic exchange owing to the lack of an available
pairing partner. Immunoblots done with extracts from the
triploid tiger lily gave the same trend as the diploid. The
greatest signal was seen in extracts from leptotene-early
zygotene, then it declined toward pachytene. We then
compared the numbers of chromosomal foci seen in our
control diploid with that seen in the triploid nuclei. At
early and mid-zygotene the triploid had somewhat more
foci than the diploid control. But by early pachytene the
103
number of foci in the diploid and triploid was very similar. Thus by the end of zygotene, the number of Dmc1
foci present is proportional to the amount of intimately
paired chromosome axis (which is the same in the diploid and triploid), and is not proportional to the total
amount of chromosome axis (which is 33% higher in the
triploid).
The diploid species-hybrid, with homeologous chromosomes, has relatively normal chromosome alignment,
an intermediate reduction in SC formation, and a large
reduction in reciprocal genetic exchange, presumably
due to a failure at the chromosome homology checking
stage. Our results with the species-hybrid suggest that
Dmc1 foci are only stabilized if chromosome homology
is found at that site. In the species-hybrid the number of
foci seen at early zygotene is similar to the number seen
in the normal diploid control, but the total number of
foci is dramatically reduced in the mid-zygotene nuclei.
By early pachytene the number of foci in the hybrid is
reduced to 45% of its early zygotene level, (and the
hybrid has only 43% as many foci as the diploid control
at the same stage). A comparable result was seen with
our immunoblots in which immunostaining was only
seen in the early zygotene extract. It appears that reduced chromosome homology leads to reduced numbers
of stabilized Dmc1 foci.
Rockmill et al. (1995) suggested that both Dmc1 protein and chromosome homology are required for axial
associations. Axial associations, in turn, are important to
the timely initiation of SC. Ultrastructural studies using
Rad51 and/or Dmc1 antibodies support this conclusion
(Anderson et al. 1997; Tarsounas et al. 1999). Our results
are in agreement with this conclusion. The reduction in
the number of Dmc1 foci in our species-hybrid is similar
to the reduction in SC formation (43%–67% reductions
in SC formation; Toledo et al. 1979, and Hasenkampf
and Shull, unpublished results). A successful homology
check appears to be needed to stabilize the Dmc1 foci at
a site, and to expedite the timely initiation of SC.
Dmc1 foci and reciprocal genetic exchange?
In the three types of lily we have examined, there is a
very good correspondence between the extent of stabilized Dmc1 foci and the extent to which intimate pairing
occurs. Thus one consequence of the homology check
appears to be the timely formation of SC. In S. cerevisiae
Rad51 and Dmc1 also both appear to be important for
achieving normal levels of reciprocal genetic exchange
(Bishop et al. 1992; Shinohara et al. 1992), and Dmc1
seems to have a role in ensuring that recombination
will occur between nonsister homologous chromatids
(Schwacha and Kleckner 1997). Given the need for the
Dmc1 protein for recombination, a successful homology
check also is likely required to allow the other DNA
interactions that give rise to reciprocal genetic exchange
between homologous chromosomes. A successful Dmc1mediated homology check might trigger chromatin
changes that would promote the ability of Rad51 to complete strand exchange between homologous chromosomes (Masson and West 2001). In this view, the primary role of Dmc1 is chromosome homology checking.
Rad51 might be blocked from engaging in interhomolog
interactions, until chromosome homology checking
has occurred. If homology checking is successful in a
specific chromosome region, then perhaps chromatin
changes would allow Rad51 to bind with accessory
proteins that could enhance the in vivo ability of Rad51
to accomplish strand exchange (Dresser et al. 1997).
We propose that in the lily the locations where DNA
interactions between homologs give rise to reciprocal genetic exchange are a subset of the sites (or might even be
different sites) than the ones that give rise to the visible
foci within which Dmc1 is functioning in homology
checking and in the initiation of SC formation. In lilies
(diploid, triploid or species-hybrid) the number of observed Dmc1 foci, even at early pachytene, is at least 10×
higher than the number of reciprocal genetic exchanges.
In the species-hybrid, the reduction in number of stabilized Dmc1 foci (45%) correlates well, and directly,
with the observed reduction in SC formation. We assume
that the extent of stabilized foci represents the extent to
which the homeologous chromosomes of the two parental species share regions of chromosome homology, and
'pass' the homology check. Based on this assumption one
might then expect Black Beauty to have 45% as many
chiasmata as the diploid control. However, chiasmata
formation in the hybrid is much more drastically reduced: Black Beauty has only ~10% of the level of reciprocal exchange seen in our normal, diploid control.
This result suggests that a further, more stringent, level
of chromosome homology is required to allow reciprocal
genetic exchange, than is needed for timely SC initiation. The higher stringency for homology might be enforced at the same site, but at a later step, downstream of
the role of Dmc1/Rad51 in the double-strand pathway.
Alternatively Dmc1-mediated homology checks might
need to be successful at several sites of a particular chromosome region (perhaps a chromosome arm) before proposed chromatin changes could occur that would promote
the ability of Rad51 to complete strand exchange between
homologous chromosomes. The higher homology demands seen for reciprocal genetic exchange might be
accomplished quantitatively by requiring that several adjacent chromosome regions all pass the homology check.
We favor this quantitative multi-locational check of homology because demanding homology at more than one
site within a chromosome region likely more accurately
measures chromosome homology than would one high
stringency test of DNA sequence complementarity at a
single site.
The pachytene checkpoint
Studies of certain types of meiotic mutants have pointed
to the existence of a pachytene checkpoint (reviewed in
104
Roeder and Bailis 2000). It is proposed that the pachytene checkpoint is activated as recombination is initiated;
once activated, nuclei cannot pass the checkpoint until
recombination is completed, and the block is deactivated. The Black Beauty hybrid proceeds from pachytene to
metaphase I, despite the fact that it has very few reciprocal genetic exchanges. If the pachytene checkpoint
model is correct, then either there is no pachytene checkpoint in the lily, or recombination is not initiated in regions that fail in homology checking, or the completion
of even a few reciprocal exchanges is enough to allow
the nucleus to pass the checkpoint.
In conclusion, we interpret our results to indicate that
the primary role of Dmc1 is homology checking. In the
lily, if the homology check is successful, then SC formation can be initiated locally, and if several successful
homology checks occur, regional chromatin events can
be triggered that would allow Rad51 to interact with the
appropriate accessory factors and promote strand exchange between homologous chromosomes. Thus our
studies support the widely held view that Dmc1 functions in homology checking. In lilies it is likely that successful homology checks promote both SC initiation and
reciprocal exchange, but that these post-homology
checking events are separable and can be regulated differentially.
Acknowledgements The authors wish to thank Dr. Tomoko
Ogawa for the generous gift of Dmc1 immune serum. They also
wish to thank Dr. Dan Riggs for assistance with the immunoblotting portion of this study. This project was supported by a Research grant to C.A.H. from the Natural Science and Engineering
Research Council of Canada, a University of Toronto fellowship
to S.G., and a research grant to K.H. from the Japanese Society for
Promotion of Science Research for the Future Program (JSPSRFTF9716001). The experiments reported here comply with the
current laws of the country in which the experiments were done.
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