Chromosoma (1999) 108:384–392
© Springer-Verlag 1999
Homolog pairing and meiotic progression in Coprinus cinereus
Libo Li, Erin E. Gerecke, Miriam E. Zolan
Department of Biology, Indiana University, 1001 East 3rd Street, Bloomington, IN 47405, USA
Received: 4 January 1999; in revised form: 22 June 1999 / Accepted: 20 July 1999
Abstract. We have used fluorescence in situ hybridiza-
tion to examine homolog pairing during the synchronous
meiosis of the basidiomycete Coprinus cinereus. Using
spread preparations of meiotic nuclei, we confirmed pre-
vious studies that showed that at 6 h post-karyogamy es-
sentially all meiotic nuclei are in pachytene. We found
that homolog pairing occurs rapidly after karyogamy,
that a 1 Mb chromosome does not associate more quick-
ly than a 2.5 Mb chromosome, and that interstitial, sin-
gle-copy sites can associate stably prior to nucleolar fu-
sion. Analysis of two probes for the same pair of homo-
logs revealed that by 4 h after karyogamy each chromo-
some examined was at least partially paired in all meiot-
ic cells. In addition, these studies showed that chromatin
condensation increases after pairing and that chromatin
shows stable compaction at pachytene.
Introduction
The function of meiosis is the precise segregation of ho-
mologous chromosomes. In order to achieve perfect seg-
regation, homologs are brought together, checked for ho-
mology, and held together as a bivalent, either by the
combined effects of recombination and sister chromatid
cohesion or by other stabilizing forces (reviewed in
Kleckner 1996; Roeder 1997; Moore and Orr-Weaver
1998; Zickler and Kleckner 1998). These associations
are maintained until anaphase I, when they are released
so that homologs may be pulled to opposite poles of the
meiosis I spindle.
Recent studies have made use of fluorescence in situ
hybridization (FISH) to examine homolog pairing in di-
verse organisms. Two main approaches have been used.
In the first, chromosome spreads are analyzed; this pro-
cedure allows the facile analysis of large numbers of
Edited by: P. Moens
Correspondence to: M.E. Zolan
e-mail: mzolan@sunflower.bio.indiana.edu
meiotic cells. The second procedure analyzes pairing
within intact nuclei and preserves spatial information
that is lost when the nuclear contents are spread onto a
surface. Both of these types of studies have yielded use-
ful information. For Saccharomyces cerevisiae, it was
found that homologs are paired prior to entry into meio-
sis (Weiner and Kleckner 1994; Kleckner 1996); this is
also true for Drosophila melanogaster, in which somatic
pairing of homologs is established by the 14-cell stage
of embryogenesis (Hiraoka et al. 1993; Fung et al.
1998). In contrast, homologs are not paired prior to mei-
osis in Zea mays (Bass et al. 1997) or in Caenorhabditis
elegans (Dernburg et al. 1998). In humans, mitotic pair-
ing of some homologous sequences has been demon-
strated (Arnoldus et al. 1989; LaSalle and Lalande
1996), but pre-meiotic pairing was not seen in spermato-
gonia (Scherthan et al. 1996).
Several investigations have examined the changes in
chromosome associations that occur at the onset of mei-
osis. In S. cerevisiae, pre-meiotic S-phase results in the
disruption of homolog pairing, which is rapidly restored
as meiosis begins (Weiner and Kleckner 1994). In both
humans and maize, it has been found that chromosomes
undergo extensive reorganization during early stages of
meiosis (Dawe et al. 1994; Scherthan et al. 1996; Bass et
al. 1997).
All of the above studies have examined meiosis in or-
ganisms that have a stable vegetative diploid form. In
these cases, the transition between mitotic and meiotic
cell cycles is purely regulatory. However, in numerous
fungi there is no vegetative diploid, and karyogamy, or
fusion of the two parental nuclei, represents the initia-
tion of meiosis. Thus, in these organisms homolog pair-
ing is a part of meiosis itself. In addition, if pre-meiotic
DNA replication precedes karyogamy, then homolog as-
sociations established during meiosis should be main-
tained without interruption until anaphase I.
The basidiomycete Coprinus cinereus grows vegeta-
tively as a stable haploid monokaryon or as a stable di-
karyon. Under appropriate conditions, the dikaryon dif-
ferentiates and forms fruiting bodies (i.e., mushrooms).

Each mushroom cap contains approximately 10 million
meiotic cells, which undergo meiosis synchronously, be-
ginning with nuclear fusion (Raju and Lu 1970; Seitz et
al. 1996). Thus, there can be no pre-meiotic homolog
pairing in C. cinereus. In order to investigate the process
of homolog pairing after karyogamy, we performed
FISH analysis on spread chromosomes at meiotic time-
points representing stages from karyogamy through
pachytene. Our results indicate a rapid association of ho-
mologs, which is maintained throughout the extensive
chromatin condensation of prophase I.
Materials and methods
Strains
All experiments were performed with two derivative isolates of
the wild-type C. cinereus strain Java-6 (J6; Binninger et al. 1987),
which had been crossed and then backcrossed four times to a sec-
ond wild-type strain, Okayama-7 (Wu et al. 1983). These isolates
(J6;5-4 and J6;5-5) were mated to one another and induced to
fruit, following a standard light and temperature regimen (16 h
light, 8 h dark at 25°C; Zolan et al. 1988).
Meiotic chromosome spreads
Meiotic tissue was isolated from mushroom caps at designated
times during meiosis, and chromosome spreads were prepared as
described (Pukkila et al. 1992; Seitz et al. 1996) using Probe-On
Plus positively charged slides (Fisher Scientific). Slides were
dried overnight at room temperature and stored at –20°C until use.
Probes
DNA probes used for FISH are described in Table 1. Probes were
isolated as overlapping cosmids from chromosome-specific librar-
ies of Okayama-7 chromosomes 8 and 13 (Zolan et al. 1992), ex-
cept cosmid 202A4, which was isolated from a total genomic cos-
mid library (May et al. 1991). To determine the complexity of the
probes, cosmid DNA was digested with restriction enzymes, sub-
jected to gel electrophoresis, and, for probes 1–5 and 7, examined
by Southern blot analysis, using the various cosmids as probes
against the other in the pair (Ramesh 1995 and data not shown).
Confirmation of the chromosomal location of probes 1–5 and 7
was obtained by using each cosmid as a probe against Southern
blots of agarose gels containing separated Okayama-7 chromo-
somes. These were obtained by contour-clamped homogeneous
electric field separation of agarose-embedded chromosomes (us-
Table 1. Probe list. (Chr. chromosome)
Probe number Chromosome DNA probe Complexity
(approx. kb)
1 8 1B11/202A4 66
2 8 2G7/3F2 61
3 13 1B2/2B9 42
4 13 1B3/3Hl2 56
5 13 1D5/3Dl2 53
6 13 1G11/1H7 36
7 13 1B2/2B9, 185
1B3/3H12,
1B10/1D9,
1D5/3D12
385
ing a BioRad CHEF DRII or CHEF Mapper; Zolan et al. 1992;
data not shown). Probe 6 represents subtelomeric DNA obtained
by performing a 32P-labeled primer extension reaction using a
42-mer [(TAACCC)7] designed to amplify from a telomeric re-
gion. The reaction consisted of 10 ng or 200 ng of Okayama-7
genomic DNA, 1×PCR buffer without MgCl2 (Fisher Scientific),
1.5 mM MgCl2, 0.5 µM of the oligonucleotide primer, 0.12 mM
dCTP/dGTP/dTTP, 5% (w/v) acetamide, 1 U Taq DNA polymer-
ase, and 50 nCi of [32P]dATP (ICN). Amplification conditions
were as follows: (1) 94°C, 4 min; (2) 92°C, 1 min; (3) 52°C,
1 min; (4) 72°C, 3 min plus 3 s per cycle (40 cycles to step 2); (5)
72°C, 6 min (final extension). The labeled product was used to
probe the chromosome 13-specific library using standard colony
hybridization procedures (Sambrook et al. 1989), as a result of
which cosmids 1G11 and 1H7 were identified and subsequently
isolated. Confirmation of their chromosomal location was ob-
tained by performing FISH using probe 6 in combination with
probe 3, which had previously been mapped to chromosome 13.
Probes for FISH were labeled using nick translation kits, in-
corporating either biotin-14-dATP (GibcoBRL Life Technologies)
or digoxygenin-11-dUTP (Boehringer Mannheim). Probes were
ethanol precipitated and stored at –20°C in 2×SSC, 50% (v/v)
formamide (Amresco), 10% (w/v) dextran sulfate, and 1 mg/ml
salmon sperm DNA. (1×SSC is 0.15 M NaCl, 0.015 M sodium ci-
trate.)
Fluorescence in situ hybridization
Hybridization and detection methods were based on published
procedures (Scherthan et al. 1992; Weiner and Kleckner 1994;
McFadden 1995), with modifications. Slides were baked at 65°C
for 2–3 h, then incubated with 100 µl of 0.1 mg/ml RNase A
(Amresco) in 2×SSC at 37°C for 30 min. Slides were washed
three times in 2×SSC for 2 min each, and dehydrated in a
70%–85%–100% ethanol series at room temperature (2 min each).
The slides were then denatured in 2×SSC containing 70% (v/v)
formamide at 70°C for 2 min, dehydrated in a cold (4°C) ethanol
series (70%–95%–100%; 2 min each), acetylated [0.1 M trieth-
anolamine-HCl (Sigma), pH 8, 0.5% (v/v) acetic anhydride
(Fisher)], and washed in 2×SSC at room temperature for 5 min.
Labeled probes (1 µg of each probe per slide) were denatured at
75°C for 5 min and added to the slides. Coverslips were added
and sealed with rubber cement. Slides were incubated in a humidi-
fied container at 37°C for approximately 36 h.
Following hybridization, slides were washed in 2×SSC con-
taining 50% (v/v) formamide at 37°C for 30 min, followed by two
10 min washes in 2×SSC at 37°C and two 10 min washes in
0.5×SSC at 50°C. Slides were then blocked with 5% (w/v) BSA
(Sigma A-3059), 0.1% (v/v) Tween-20 in 4×SSC at room temper-
ature for 10 min. Detection of the probes involved incubating
slides in 5 µg/ml UltraAvidin-rhodamine (Leinco Technologies) at
37°C for 30 min, washing three times in 4×SSC, 0.1% (v/v) Triton
Source References
Chr. 8/9-specific cosmid library Zolan et al. (1992),
Ramesh (1995)
Chr. 8/9-specific cosmid library Ramesh (1995)
Chr. 13-specific cosmid library This paper
Chr. 13-specific cosmid library This paper
Chr. 13-specific cosmid library This paper
Chr. 13-specific cosmid library This paper
Chr. 13-specific cosmid library This paper
386

X-100 at 37°C for up to 10 min each wash, incubating in 5% (v/v)

goat serum (Vector Laboratories) in 2×SSC at room temperature
for 5 min, incubating in 5 µg/µl biotinylated anti-avidin D (Vector
Laboratories) and 10 ng/µl antidigoxygenin-fluorescein Fab frag-
ments (Boehringer Mannheim) at 37°C for 30 min, and washing
in 4×SSC, Triton X-100 at 37°C. Slides were then blocked in 5%
BSA, 0.1% Tween-20 in 4×SSC at room temperature for 5 min,
incubated in 5 µg/ml UltraAvidin-rhodamine and 10 ng/µl anti-flu-
orescein antibodies (Boehringer Mannheim) at 37°C for 30 min,
washed three times in 4×SSC, Triton X-100 at 37°C, incubated
with FITC-conjugated rabbit anti-mouse immunoglobulins (1 in
50 dilution of stock in 5% BSA, 0.1% Tween-20 in 4×SSC;
DAKO) at 37°C for 30 min, and finally washed with 4×SSC, Tri-
ton X-100 at 37°C. Slides were mounted in Vectashield + 4’,6-di-
amidino-2-phenylindole (DAPI) (Vector Laboratories) to counter-
stain chromosomes and prevent fading, coverslips were added and
sealed with nail polish, and slides were stored in the dark at 4°C
for up to several weeks.

Microscopy and data analysis

Nuclei were examined using a PlanApo 60× objective on a Nikon

Microphot-FXA fluorescence microscope equipped with a filter
wheel (Ludl) containing a Chroma technology 8300 filter set. Im-
ages were captured and stored using a Photometrics cooled digital
CCD camera system and Quips XL SmartCapture/IP Lab Spec-
trum FISH imaging software (Vysis). Distances between foci of
probe hybridization were measured using the same imaging soft-
ware (see Fig. 2A). Data were recorded and analyzed using Mi-
crosoft Excel 4.0.

Results

FISH analysis and meiotic progression during prophase I

In order to examine meiotic homolog pairing in C. cine-

reus, we prepared chromosome spreads of nuclei at vari-
ous meiotic timepoints and analyzed them using FISH.
The probes we chose (Table 1) ranged in complexity
from approximately 36 to 185 kb. In most experiments,
we used a probe complexity of about 50 kb, which con-
sisted of two overlapping cosmid clones. In previous
studies (Ramesh and Zolan 1995; Seitz et al. 1996), we
defined karyogamy (K) as the time at which roughly
50% of meiotic cells contain single nuclei; in our experi-
ence, this occurs approximately 1 h before the lights
come on when cultures are maintained under a 16 h light,
8 h dark regimen. For the experiments shown in this pa-
per, we analyzed homolog associations in spreads of
fused or fusing nuclei in which the two parental nucleoli
were either unfused (Fig. 1A) or fused (Fig. 1B–D).
Thus, an experiment at a K timepoint would be an analy-
sis of those 50% of meiotic nuclei that had fused.
As first shown by Raju and Lu (1970) and subse-
quently by numerous studies (e.g., Seitz et al. 1996), Fig. 1A–D. Time-course of chromatin compaction and homolog
meiosis progresses synchronously in C. cinereus. Meiotic pairing in Coprinus cinereus. Chromosomes were spread as previ-
stage variations are most noticeable among nuclei spread ously described (Pukkila et al. 1992; Seitz et al. 1996) and hybrid-
at and near K (Table 2), because the early stages of mei- ized with probes for chromosomes 8 (green) and 13 (red), as de-
scribed in the text. Panels A and B are spreads from Experiment
otic prophase are relatively short. In contrast, at 6 h after 1, panel C is from Experiment 10, and panel D is from Experi-
K (K+6) essentially all nuclei (97%; Table 2) are in ment 12 (Table 3). Arrows in panels A and B denote nucleolar re-
pachytene. Using spreads from K+6 nuclei, we exam- gions remaining after RNase treatment. Bar represents 1 µm
ined the distances between foci representing allelic sites
for chromosomes 8 and 13 (green to green and red to red
signal distances, respectively; Fig. 2A), and we also
 387

Fig. 2A–D. Distances between homologous and nonhomologous
foci for chromosomes 8 and 13. A Focal distance measurement
technique. Homologous chromosome focal distances were mea-
sured between foci of the same color (H 1 or H 2). Nonhomolo-
gous distances were measured between foci of different colors
(NH 1–NH 4). For a given nucleus, one or two foci were observed
for each probe. If a single focus was observed, the homologous
distance was recorded as 0 µm. One to four nonhomologous dis-
tances were recorded for each nucleus, based on the number (2 to
4) of homologous foci observed. B Meiotic nuclei at 6 h after
karyogamy (K+6; Table 3, Experiments 12, 13, and 14). Measure-
ments for each nucleus were plotted in increasing order, based on
the smallest NH distance for that spread. The scale for this plot
was chosen to facilitate comparison with panel D. For eight nu-
clei, the minimum NH distance exceeded 15 µm; NH distances for
these samples are therefore not shown (the maximum NH distance
observed for any nucleus was 32 µm). C Same data as in B, ex-
cept that focal distances of 3 µm or less only are shown. For this
panel, measurements are plotted as increasing values for each type
of measurement (H 1, H 2, etc.; as in Weiner and Kleckner 1994).
Therefore, the set of data for each nucleus number does not reflect
the measurements derived from a single nucleus. D Nuclei from
Experiment 1 (Table 3). Plot symbols for panels B–D: black cir-
cles chromosome 8 homologous distance (H 1), gray circles chro-
mosome 13 homologous distance (H 2), plusses nonhomologous
distance 1 (NH 1), hollow squares NH 2, hollow circles NH 3,
hollow triangles NH 4. In cases in which only one focus was pres-
ent for a given probe, fewer than four NH distances were mea-
sured; therefore, plots for NH distances 3 and 4 usually have few-
er points than those for NH distances 1 and 2

Table 2. Percentage of nuclei at various meiotic stages casionally (especially in uncondensed nuclei), more than
two signals would be apparent for a given probe; one or
Meiotic stagea more of these signals were usually about half the intensi-
Timepoint A B C D Nb ty of a normal focus, and these smaller signals tended to
cluster together. These smaller signals could represent
K–1 39 43 13 5 186 the sister chromatids comprising a given homolog, or
K 24 29 9 39 114 they could reflect the decondensed state of the chroma-
K+l 1 23 8 67 243 tin. Alternatively, they could result from increased
K+4 10 4 86 153 spreading forces acting on a given nucleus.
K+5 3 6 92 120
K+6 1 2 97 487 Distances were measured linearly between all combi-
nations of signals present; thus, for a given nucleus, one
a Percent nuclei at the meiotic stages shown in Fig. 1 to four nonhomologous distances could be recorded, de-
b Number of nuclei from all experiments shown in Tables 3–5 pending on the number of individual allelic foci present.
Figure 2B presents a graphical depiction of data ob-
tained from nuclei at K+6 (Table 3, Experiments 12–14).
measured the distances between nonhomologous signals The distances measured for each nucleus were plotted at
(red to green distances; Fig. 2A). For a given probe, the the same ordinate of the x-axis, and the data were ar-
distance between signals was measured as the distance rayed in increasing order by the minimum nonhomolo-
from the center of one focus to the center of the other. gous distance measured for that nucleus. Our analysis
Usually, one or two signals per probe were visible. Oc- revealed that, for each probe, allelic foci were associated
388

Table 3. Pairing of chromosomes 8 and 13 at various meiotic timepoints. (Chr. chromosome)

Timepoint Expt.a Chr. 8 probeb Chr. 13 probeb NB–Dd Percent pairing

Chr. 8 Chr. 13 Both chr.8 Either chr.8

AND chr. 13 OR chr. 13

K–1e 1 2 3 30 93 87 83 97
2 2 3 36 83 86 78 92
K 3 2 3 26 92 96 92 96
4 2 3 18 83 78 78 83
5 2 3 8 100 100 100 100
K+l 6 1 5 95 94 96 91 99
K+4 7 2 4 15 100 100 100 100
8 2 5 46 98 96 93 100
9 2 5 27 93 96 93 96
K+5 10 1 3 104 93 96 89 100
11 1 5 16 88 88 75 100
K+6 12 1 3 108 96 100 96 100
13 2 5 13 100 100 100 100
14 2 5 12 100 92 92 100
15 2 6 107 95 99 94 100
16 – 7 57 – 100 N.A. N.A.
a Experiment. This means spreads from one mushroom, and one d Refers to meiotic stages represented by nuclei in Fig. 1; all pair-
hybridization. Up to three slides may be included per experiment ing calculations are based on nuclei at these stages
b Probe descriptions are in Table 1 e At K–1, only Fig. 1B-type spreads are included in the analysis;
c Number of nuclei per experiment see text

at a distance of 1.1 µm or less in more than 90% of these
nuclei. We also plotted each set of measurements inde-
pendently in increasing order (as in Weiner and
Kleckner 1994; Fig. 2C). This analysis revealed that
the distribution of interallelic distances is continuous to
1.1 µm; therefore, we defined this distance as the limit at
which foci would be designated as paired.
Using an interallelic distance of 1.1 µm or less as the
criterion for pairing, we examined nuclear spreads in
which condensed, aligned homologs were not apparent,
and found that homolog pairing occurs rapidly after nu-
clear fusion. For the earliest timepoint examined (K–1),
we analyzed only spreads of the type shown in Fig. 1B,
in order to examine a more homogeneous population of
nuclei just after nucleolar fusion (Table 3, Experiments 1
and 2). In these experiments, the range of pairing seen
for an individual probe was 83%–93%. We found that
chromosome 13, which is 1 Mb in the strain used (as de-
termined by pulsed-field gel electrophoresis; data not
shown), is no more likely to be paired than chromosome
8, which is approximately 2.5 Mb in size. In both experi-
ments at K–1, more than 90% of the nuclei were paired
for at least one of the two sites probed, indicating that
some homolog association was present in most cells.
However, fewer of these cells showed pairing for both
sites assayed (for example, 97% vs 83% for Experiment
1; Table 3). Therefore, homolog pairing is likely incom-
plete in nuclei from mushrooms at early meiotic time-
points. A plot of the pairing data from Experiment 1
(Fig. 2D) demonstrates the greater range of allelic dis-
tances measured for spreads from these nuclei.
For pachytene nuclei, pairing associations detected by
FISH analysis are easily confirmed by direct observation
of the stained chromosomes (Fig. 1D). However, for the
diffuse nuclei seen earlier in meiosis (Fig. 1A, B), esti-
mates of pairing are dependent solely upon the analysis of
fluorescent signals. Nonspecific background signals could
result in the appearance of less pairing than truly exists for
a given probe (if two spots are observed on a nuclear
spread instead of one). Conversely, if one of two signals
for a given probe is lost the locus may be misidentified as
paired. To investigate the possibility that our estimates of
pairing in early meiotic nuclei were inflated by the latter
technical artifact, we examined mitotic nuclei present on
the slide used for Experiment 2 (Table 3). Mitotic nuclei
are distinguished by their smaller size and smaller nucleo-
li. We found for each probe that signal was present in 73%
of the 30 nuclei examined. In the meiotic cells of the same
slide, the chromosome 8 probe was scored as paired in 31
of 36 nuclei. Of these, 9 had two distinct signals and were
therefore not subject to the possible artifact of signal loss.
The remaining 21 spreads showed one signal for the chro-
mosome 8 probe (we conservatively scored it as one signal
even if the spot was broad, as if resulting from two inde-
pendent hybridization events). If we apply a correction
factor of 0.73 to this class of nuclei, the total percent pair-
ing for chromosome 8 was 67% (9+15/36). By a parallel
analysis, the total percent pairing for chromosome 13 in
the same experiment was 69%. Thus, there is a possible in-
flation of our pairing estimates (e.g., 67% vs 83% for chro-
mosome 8) reported for early timepoints in Table 3. How-
ever, even by our most conservative calculations, we con-
clude that homolog pairing is rapid after nuclear fusion.
A slide prepared from a mushroom at an early meiot-
ic timepoint will contain nuclear spreads showing vari-
able levels of chromatin compaction (Fig. 1; Table 2). In
 389

Table 4. Pairing at two different sites on a single chromosome. (Chr. chromosome)

Chr. Time Expt. Probe aa Probe b Nb Percent pairing

point (biotin) (dig)
Probe a Probe b Both probe a Either probe a
AND probe b OR probe b

8 K–1 17a 1 2 47c 34 30 15 49

8 K–1 17b 1 2 67 87 87 80 94
8 K 18 1 2 35 91 94 89 97
8 K+4 19 2 1 65 97 95 92 100
8 K+6 20 2 1 12 75 100 75 100
8 K+6 21 1 2 58 97 98 95 100
13 K+1 22 6 3 145 91 81 79 93
13 K+6 23 6 3 120 100 98 98 100
a Probe descriptions are in Table 1
b Number of nuclei examined in stages B–D (Fig. 1), except where noted
c Nuclei for this experiment are from stage A (Fig. 1)

our spreads, nuclei of the type shown in Fig. 1C were al-
ways a small component of the nuclei (Table 2; Seitz et
al. 1996). This is in contrast to other studies of C. cine-
reus (e.g., Lu and Raju 1970), in which considerable
chromosome condensation was observed, even before
karyogamy; we do not know the basis for this difference.
In Fig. 1C, unpaired chromosome segments may be
present, and these segments could reflect incomplete
pairing associations within the nucleus and/or the dis-
ruption of chromosome pairing when nuclei of this type
are spread. However, our measurements of percent pair-
ing for individual probes did not decrease as chromatin
became more compact; for example, in Experiment 1,
pairing for the chromosome 8 probe was 93% for the 30
spreads of the Fig. 1B type included in Table 3, 92% for
12 spreads of the Fig. 1C type observed, and 100% for
the 5 detected spreads of the Fig. 1D type. This indicates
that once homologs associate in C. cinereus, they most
likely do not lose this association prior to synaptonemal
complex formation, at pachytene.
Traditionally, nucleolar fusion defines the beginning of
meiosis in C. cinereus (Raju and Lu 1970). However, we
found that homolog associations were apparent prior to nu-
cleolar fusion. We examined 49 spreads of nuclei with un-
fused nucleoli (Fig. 1A) from caps at the K–1 and K time-
points, using interstitial probes for chromosomes 8 and 13.
For each probe examined, about 40% of the nuclei showed
pairing. Both sites were paired in 24% of the nuclei, and at
least one site showed pairing in 68% of the nuclei. This
greater contrast between the percent of nuclei with some
pairing and those with both sites paired means that homo-
log associations stable to our spreading technique are in
the process of forming at this stage of meiosis.
Pairing of two sites on the same chromosome
The analysis of a single site on each chromosome will
underestimate the extent of pairing of that chromosome,
according to models of S. cerevisiae homolog associa-
tion early in meiosis (Weiner and Kleckner 1994;
Kleckner 1996). If homolog associations are initially rel-
atively unstable, then a certain amount of “breathing,” or
opening, is expected between specific sites on chromo-
somes, even if homologs are completely aligned. Thus, a
given site may be more than 1.1 µm away from its allelic
site even though these homologs are paired overall. To
examine this possibility, we probed either two sites for
chromosome 8 or two sites for chromosome 13 in the
same experiment (Table 4). Both of the chromosome 8
sites were interstitial, as was one of the chromosome 13
sites. The second chromosome 13 probe (probe 6, Table 1)
consisted of overlapping cosmids that were isolated by
hybridizing a chromosome 13-specific cosmid library
with a probe made by primer extension from an oligonu-
cleotide to the presumptive telomeric repeat region
(Materials and methods). This probe consists of single-
copy DNA, and it hybridizes specifically to one end of
chromosome 13 (data not shown).
We analyzed two sites on chromosome 8 in nuclei
from early timepoints. In spreads with unfused nucleoli,
each probe revealed about 30% pairing (Table 4, Experi-
ment 17a), consistent with results described above for ex-
periments in which one probe for each of two chromo-
somes was used. However, 49% of these nuclei show as-
sociation of at least one site. After nucleolar fusion, nearly
all nuclei show association for at least one site on chro-
mosome 8 (Table 4, Experiment 17b); a similar result was
found for the chromosome 13 analysis (Table 4, Experi-
ment 22). Therefore, experiments in which single sites are
measured for each chromosome (Table 3) may underesti-
mate the true amount of homolog association present.
Chromatin compaction after pairing
The use of two probes for the same chromosome also al-
lowed us to examine the relationship between chromo-
some compaction and pairing in C. cinereus. Although
one study in S. cerevisiae found that compaction pre-
ceded pairing (Scherthan et al. 1992), another analysis
(Weiner and Kleckner 1994) found that compaction in-
creased after at least one site on a pair of homologs had
paired. We analyzed nuclei from K–1, K, K+4, and K+6
timepoints and used only those spreads in which both
sites examined on chromosome 8 were paired (Table 5).
390

Table 5. Compaction of chromosome 8

Stagea Time Expt.b Nc Distance between probes (µm)
point
Min. Max. Mean SD Median

B K–1 17b 40 0.31 7.03 2.99 1.52 2.79

B K 18 14 0.38 8.55 3.21 1.95 2.50
D K 18 15 0.79 5.93 2.24 1.08 2.12
D K+4 19 45 0.92 5.11 2.81 0.83 2.75
D K+6 20 11 1.54 4.23 2.60 0.72 2.37
D K+6 21 57 1.58 5.25 2.66 0.57 2.55
a Refers to meiotic stage of the nuclei; see Fig. 1
b Probes as in Table 4
c Number of nuclei scored per experiment. For nuclei in stage B, N includes only those nu-
clei for which both probes were paired. For nuclei in stage D, all nuclei were scored

Discussion

Recent studies of meiotic homolog pairing have utilized

Fig. 3. Distances between two foci on the same chromosome. Dis-
tances are plotted for stage B nuclei from Experiment 17b (Table
5; circles) and stage D nuclei from Experiment 21 (Table 5; trian-
gles). For comparison, the smallest NH distances from Fig. 2D
(plusses) are also shown
For each spread, we measured the distance between the
signals of the two loci probed. When a locus is paired, it
is detected as either two closely spaced spots of fluores-
cence or a single spot; therefore, one to four distances
were measured per nucleus (Fig. 2A; NH distances). We
compared spreads of types B and D (Fig. 1) in order to
compare pachytene with earlier nuclei that have relative-
ly diffuse chromosomes. Compared with the pachytene
spreads, the diffuse nuclei showed a greater range of dis-
tances and a larger average distance between probe sig-
nals (Table 5). When chromosomes are diffuse, they are
so flexible that the distances between two measured
points are essentially random (Fig. 3); they have the
same distribution as that of distances between foci on
nonhomologous chromosomes. In contrast, at K+6 the
pachytene chromosomes are compact and less flexible;
hence, there is less variability in the distance between
two chromosomal loci (Fig. 3; Table 5). Thus, there is
compaction after homolog pairing in C. cinereus. Be-
cause we do not know the physical distances between
probes, we cannot say how much compaction is present
in the diffuse spreads. However, based on the appear-
ance of meiotic chromatin in a cell undergoing karyoga-
my (especially see Raju and Lu 1970), it is likely that
some compaction has already taken place.
FISH analysis of chromosome spreads (e.g., Loidl et al.
1994; Weiner and Kleckner 1994) or intact nuclei (Bass
et al. 1997; Dernburg et al. 1998) in order to examine
the transition of diploid cells from mitosis to meiosis.
This study represents the first FISH-based examination
of homolog pairing in a zygotic meiosis without an ex-
tensive preceding diplophase. Because additional studies
will concern the examination of homolog pairing in mei-
otic mutants in which chromatin is never completely
condensed (Ramesh and Zolan 1995; Seitz et al. 1996;
E.E.G. and M.E.Z., manuscript submitted), we preferred
the approach of using relatively small (30–50 kb)
probes, rather than chromosome painting. The painting
approach has been used to examine homolog pairing in
wild-type and mutant strains of yeast (Scherthan et al.
1992; Loidl et al. 1994; Nag et al. 1995), but uncon-
densed nuclei had to be excluded from examination.
We found that homolog pairing is rapid in fusing nu-
clei; a substantial proportion of cells exhibited pairing
of allelic sites even before nucleolar fusion (Table 4,
Experiment 17a). Kanda et al. (1990) found that pre-
meiotic DNA replication occurs prior to karyogamy in
C. cinereus. Their results imply that homolog associa-
tions established during meiosis should persist uninter-
rupted until anaphase. Our data show that this is in fact
the case: the proportion of cells showing pairing of inter-
stitial sites increased between the K and K+6 timepoints
(Table 3). We also found that spreads in which little
chromatin condensation was apparent (Fig. 1B) and
those in which threadlike chromosomes were visible
(Fig. 1C) showed high levels of homolog association.
This confirms results from three-dimensional recon-
struction of meiotic nuclei (of Sordaria macrospora;
Rhoades 1961; Zickler and Kleckner 1998), and gives
further experimental support to the notion (Weiner and
Kleckner 1994) that the chromosomes in these apparent-
ly disordered arrays are already associated as homologs.
The analysis of single probes for two different chro-
mosomes (Table 3) did not permit us to distinguish be-
tween homolog pairing (i.e., alignment) and homolog co-
localization in a looser sense (Weiner and Kleckner 1994;
Kleckner 1996). However, the analysis of two sites on a

single chromosome (Table 4) revealed that both sites are
paired in the large majority of nuclei in which nucleoli
have fused. Since the sites used were separated by more
than three-quarters (for chromosome 8) or one-half (for
chromosome 13) of the length of the homologs involved,
it is likely that these associations are between aligned
chromosomes, and that most sites along them are paired.
This analysis also did not permit us to distinguish di-
rectly between homolog pairing and synapsis. It is
known that C. cinereus undergoes complete axial core
development before synapsis (tripartite synaptonemal
complex formation; Holm et al. 1981; Seitz et al. 1996)
and that it displays presynaptic alignment (Lu 1967; Lu
and Raju 1970; Holm et al. 1981). Therefore, it is most
likely that homolog pairing, as assayed by FISH, would
precede synaptonemal complex formation. Our studies
fully support this idea: pairing of homologs occurs im-
mediately after karyogamy, even before axial core for-
mation is complete (Holm et al. 1981; Seitz et al. 1996).
Our studies also showed that there is chromatin com-
paction after pairing, but we were not able to determine
its extent from our data. Because we examined two-di-
mensional spreads, we could not follow the length of in-
dividual chromatids in nuclei from early timepoints.
Therefore, our measured value for the distance between
two foci for spreads of the Fig. 1B type will often be an
underestimate. Conversely, the relatively uncondensed
chromatin of early timepoints will also result in spreads
in which two loci along a given chromatid lie artificially
close to each other, due to folding of an unsynapsed uni-
valent. Overall, the measured distance between probes on
a given chromosome will show a wider range when mul-
tiple nuclei are compared, and the variance for that popu-
lation will be much higher than that for pachytene nuclei,
in which chromosomes are condensed to a standard, rela-
tively uniform length (Table 5; see also Pukkila and Lu
1985; Seitz et al. 1996). A full understanding of the ex-
tent of compaction after pairing will require the analysis
of early meiotic nuclei using methods that maintain the
three-dimensional architecture of the nucleus.
Since this analysis employed surface spreads of mei-
otic nuclei, the pairing we observed must be, by defini-
tion, stable to the spreading forces, and it is formally
possible that very early homolog associations are present
before they are stable to our technique. In addition, the
geometry of the fusion of the two parental nuclei of the
dikaryon cannot be observed by our method. Therefore,
we cannot tell whether the pairing process begins after
nuclear fusion or whether homologs are in fact colocal-
ized by the act of fusion itself. This type of question
could be addressed using recently described approaches
(Dernburg and Sedat 1998) that allow the analysis of
pairing in intact cells.
Acknowledgements. We thank members of the Zolan laboratory for
helpful discussion, and Sonia Acharya, Martina Celerin, Jason
Cummings, Sandra Merino, Patricia Pukkila, and the reviewers for
critical comments on the manuscript. We are grateful to Geoffrey
McFadden for advice about in situ hybridization. This work was
supported by NIH grant GM43930 (to M.E.Z.). E.E.G. was sup-
ported by NIH Training Grant 2T32GM07757 (to the Department
of Biology, Indiana University).
391
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