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Spectral karyotyping (SKY) of mouse meiotic

chromosomes
Henry H.Q. Heng, Guo Liu, Wei Lu, Steve Bremer, Christine J. Ye, Mark Hughes,
and Peter Moens

Abstract: The spectral karyotyping procedure of in situ hybridization with chromosome-specific probes assigns a
unique colour code to each of the 21 mouse mitotic chromosomes. We have adapted this procedure to meiotic prophase
chromosomes, and the results show that each of the pachytene or metaphase I bivalents can be identified. This technique has the potential to recognize synaptic anomalies and chromosome-specific structural and behavioural characteristics. We confirm these potentials by the recognition of the heterologous synapsis of the X and Y chromosomes and by
the variances of synaptonemal complex lengths for each of the colour-coded bivalents in eight prophase nuclei.
Key words: SKY, meiosis, synaptonemal complex, multicolour, chromosome painting, spectral karyotyping, proteinSKY
co-detection.
Rsum : Le caryotypage spectral, cest--dire une hybridation in situ laide de sondes spcifiques de chaque chromosome, permet dassigner une couleur chacun des 21 chromosomes mitotiques chez la souris. Les auteurs ont modifi cette technique pour ltude de chromosomes en prophase miotique, et les rsultats indiquent que chacun des
bivalents en pachytne ou en mtaphase I peut tre identifi. Cette technique offrirait la possibilit dobserver des anomalies synaptiques ainsi que des caractristiques structurales ou des comportements qui sont spcifiques dun chromosome prcis. Les auteurs ont confirm ces possibilits en visualisant les synapses htrologues entre chromosomes X et
Y de mme quen observant la variance quant la longueur des complexes synaptonmiques pour chaque paire de bivalents (distingus en fonction de leur couleur) au sein de huit noyaux en prophase.
Mots cls : SKY, miose, complexe synaptonmique, multicolore, coloration chromosomique, caryotypage spectral, codtection protineSKY.
[Traduit par la Rdaction]

Heng et al.

298

Introduction
At prophase of meiosis, the chromosomes acquire meiosisspecific structures and behaviours that function in homologous
synapsis, recombination, and segregation. Experimentally induced defects in one or more factors that regulate meiosis in
the mouse, such as DMC1/ (Pittman et al. 1998), SCP3/
(Yuan et al. 2000), SPO11/ (Baudat et al. 2000), and
ATM/ (Barlow et al. 1998), have been reported to affect
synapsis. If there is interference with the induction of DNA
breaks (SPO11/) or the homology search mechanism is
compromised (DMC1/), it can be expected that synapsis
of homologous chromosomes will be reduced or absent.
However, significant numbers of synaptonemal complexes
(SCs) have been observed in such cases. The SCs may be
produced in spite of reduced homology recognition

mechanisms or alternately they involve nonhomologous associations.

To differentiate between these alternatives, we explore the
possibility of adapting the spectral karyotyping (SKY)
multicolour technique, developed for colour coding mitotic
chromosomes in human or mouse (Liyanage et al. 1996), to
the colour coding of mouse meiotic prophase chromosomes.
Modifications are necessary to accommodate the meiotic
specific conditions that include the lesser degree of
chromatin compaction and the simultaneous visualization of
the chromosome cores and SCs.
We show that the SKY procedure can effectively be used
to differentiate each of the meiotic prophase chromosomes
and their SCs. The detection of nonhomologous association
is demonstrated by the two-colour signal of the XY
chromosome pair, and the identification of individual chromo-

Received December 1, 2000. Accepted February 9, 2001. Published on the NRC Research Press Web site March 23, 2001.
Corresponding Editor: T. Schwarzacher.
H.H.Q. Heng,1 G. Liu, W. Lu, S. Bremer, and M. Hughes. Center for Molecular Medicine and Genetics, Department of
Pathology, Karmanos Cancer Institute, 5047 Gullen Mall, 5107 Biological Science Building, Wayne State University School of
Medicine, Detroit, MI 48202, U.S.A.
C.J. Ye. SeeDNA Biotech Inc., Windsor, ON, Canada.
P. Moens. Department of Biology, York University, Toronto, ON M3J 1P3, Canada.
1

Corresponding author (e-mail: hheng@cmb.biosci.wayne.edu).

Genome 44: 293298 (2001)

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294

somes is explored in a comparative analysis of eight complete

prophase nuclei.

Genome Vol. 44, 2001

Denaturation

Materials and methods

Slides were denatured using 70% formamide 2 SSC at 72C

for 90 s then immediately immersed for 2 min each in cold 70%,
80%, and 100% ethanol. The SKYpaint mixture was denatured at
80C for 7 min following incubation at 37C for 1 h.

Meiotic chromosome preparation

Hybridization

Testicular cells were obtained from 3- to 6-month-old mice.

Methanol acetic acid fixation

Tubular fragments from a macerated testis in minimal essential
medium (MEM) were allowed to settle in an Eppendorf tube. The
cell suspension was removed to a clean Eppendorf tube, and the
cells were pelleted by centrifugation at 1500 rpm for 3 min. Cells
were collected, hypotonically treated with 0.4% KCl for 10 min,
and fixed in either 3:1 or 2:1 methanol acetic acid. After three
changes of fixative, the chromosome preparations were made by
dropping the cell suspension onto a cold slide, which was then air
dried (Heng and Tsui 1993). Slides were baked for a few hours
then used immediately for SKY.

Ten microlitres of denatured SKYpaint was loaded on the denatured slide. After sealing the edges of the cover slip with rubber
cement, slides were transferred to a humidified chamber and incubated at 37C for 4896 h.

Detection
Slides were washed with 50% formamide in 2 SSC for 5 min,
1 SSC for 2 5 min, and then 4 SSC with 0.1% Tween 20 for
2 min. Signals were detected by following the instructions of the
detection kit from Applied Spectral Imagining Inc. The slides were
washed, air dried, stained with 4,6-diamino-2-phenylindole
(DAPI), and mounted with antifade.

Image acquisition and analysis

Paraformaldehyde fixation
Following cell preparation as indicated in the previous section,
the MEM was drained from the Eppendorf tube following
centrifugation, and the cells were gently resuspended in the residual liquid. Five microlitres of this suspension was touched to the
surface of a bath containing 0.5% NaCl, pH 8, and the nuclei were
collected on a clean glass slide. The slides were fixed first in
paraformaldehyde with 0.03% SDS at a pH of 8.2; then in
paraformaldehyde, pH 8.2, followed by three washes in 0.4% Kodak Photo-Flo 200, pH 8, for 1 min each. Slides were briefly air
dried and used immediately for SKY or stored at 20C for later use
(Heng et al. 1994, 2000).

Immunostaining of the centromeres and synaptonemal

complexes
Slides with paraformaldehyde-fixed nuclei were incubated in
three changes of blocking buffer (PBS with 10% v/v antibody dilution buffer (ADB: 10% goat serum, 3% BSA, 0.05% Triton X-100
in PBS), and 0.4% Kodak Photo-Flo) for 10 min each. The second
wash also contained 0.01% Triton X-100. Appropriate dilutions of
primary antibody were added to the slides (in this case, anti-COR1
and anti-SYN1 and the anticentromeric serum, CREST) which
were incubated at 37C for 2 h or overnight at room temperature
(Dobson et al. 1994). Following washes in a blocking buffer series
as described for primary antibody treatment, the nuclei were incubated in appropriate dilutions of the pertinent secondary antibodies
tagged with a variety of fluorochromes or gold particles. To compare the results of different combinations of SC detection and SKY
painting, three different secondary antibody tags (fluorescein
isothiocyanate (FITC), rhodamine, and gold staining) were used to
stain the SC. Following three 10-min washes in PBS with 0.4%
Photo-Flo and two 1-min rinses in distilled water with 0.4% PhotoFlo, the slides were dried and were ready for SKY detection.

SKY detection
Slide treatment
Methanol acetic acid fixed slides and whole-mount surface
spreads with paraformaldehyde-fixed meiotic chromosomes, some
of which were prestained with antibody and fluorochromes, were
used for comparison. Slides were incubated at 37C in pepsin solution (0.025 g/mL) for 1 min. After two washings with 1 PBS, followed by 1 PBS containing 50 mM MgCl, the slides were fixed
with 1% formaldehyde for 10 min. Slides were then dehydrated
with ethanol (70%, 90%, and 100%) and air dried following an additional wash in 1 PBS.

The protocol for the SKY image was followed to acquire standard SKY images. We do not recommend the standard SKY imaging procedure for image acquisition of SCs and other protein
markers such as centromeres. Although some well-spread meiotic
figures could be used to obtain chromosome contours with the
standard SKY program, we elected to utilize colour images taken
with the SKY filter only rather than the DAPI filter in order to obtain better images of the SC. Specifically, after taking a colour
photograph of the protein-stained image, a second photograph in
black and white was taken using the DAPI option for picture acquisition, but the filter was not changed to DAPI as advised for
standard SKY images. Thus the same filter is used for both the
DAPI and the spectral colour image. The SCs were stained by antiCOR1 and detected by FITC. The lengths of SCs for each spread
were measured on the computer screen.

Results
SKY chromosome painting of meiotic prophase nuclei
The appearance of SKY staining of relatively undisturbed
meiotic prophase nuclei is demonstrated in Fig. 1. The
CREST anti-centromere immune staining serves as a guide
to the early developmental stages. At the leptotene stage
where most of the chromosomes and the 40 centromeres are
not paired (Figs. 1a and 1f), the colours are relatively diffuse, indicating that no sharply defined domains are as yet
established. Somewhat later (Figs. 1b and 1g), there are
fewer centromeric signals as a result of chromosome
synapsis. The chromosome domains are somewhat more
clearly defined than at leptotene. At early pachytene
(Figs. 1c and 1h) the individual bivalents have become more
distinct and they are well defined at late pachytene where the
chromosomes are compacted and relatively short (Figs. 1d
and 1i). The effectiveness of the SKY procedure at meiosis
is demonstrated in Figs. 1e and 1f where the SKY program
can identify each bivalent by its specific colour.
SKY identification of individual pachytene
chromosomes
In whole-mount testicular cell preparations with wellspread pachytene chromosomes, each bivalent has a specific
colour (Fig. 2a). The SCs and associated chromatin are
shown in Fig. 2b. The colours of Fig. 2a are interpreted by
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Heng et al.

295
Fig. 1. Multicolour appearances of intact meiotic prophase nuclei
(a to e) and the corresponding centromere and (or) chromatin
images (f to j). (a and f) An early stage prior to the pairing of
chromosomes with approximately 40 centromeric signals that are
typically fairly evenly distributed through the nuclear volume.
The individual chromosomes do not have well-defined boundaries at this stage. (b and g) The reduction in numbers of
centromeric signals suggests that chromosome synapsis is in
progress. The chromosome domains have become more distinct.
(c and h) At early pachytene, the synapsis is complete and the
chromosome domains are well delineated. (d and i) At late
pachytene, the chromosomes are more compacted and the individual domains are well separated. (e and j) A typical metaphase
1 configuration where the colour coding of the individual bivalents is evident. The nuclei are about 40 m in diameter.

Fig. 3a, which is compared with the chromosomes or SCs of

a second nucleus in Fig. 3b. It is evident from this and from
six additional nuclei that SKY-painted mouse pachytene
karyotypes do not consistently agree with the order defined
by the mitotic chromosomes (Table 1; Fig. 3). The artefactual
and possible real causes of the discrepancies are discussed below.
Technical aspects
The SKY program was designed for the analysis of condensed mitotic chromosomes, and some modifications had to
be introduced to successfully analyse the less condensed,
more diffuse meiotic prophase chromosomes. The methanol
fixation that is routinely used for mitotic chromosome preparations proved to be less effective for pachytene chromosomes. Instead, the paraformaldehyde fixation that is
normally used for meiotic chromosome spreads and for EM
and immune staining gave better SKY-staining results. In addition, it was found that extended hybridization times improved the SKY image.
The fluorochromes used for immune staining of chromosome-associated proteins such as the chromosome cores and
the centromeres were found to be a source of optical interference with the SKY-view analysis. Comparison of FITC,
rhodamine, and gold-stained proteins indicated that FITC
produced the least interference. In addition, the dosage of
the fluorochromes had to be kept low to minimize interference.

Discussion

the SKY program and then expressed in standardized colour-coded chromosome images (Fig. 2c). The SKY images
and the SCs of Fig. 2c are presented in a sorted karyotype in

There are numerous causes for synaptic failure and the

mispairing of chromosomes at meiotic prophase as referenced in the introduction. If the effects of mutations or metabolites on chromosome behaviour at meiosis are to be
evaluated beyond the generality of synaptic defects, it is
required, among others, to know to what extent homology
recognition is reduced or abolished. Cytogenetically, synaptic failure has been deduced from the presence of univalents
at late stages of meiotic prophase, or at early prophase
stages, by observations on partially synapsed chromosomes
or by partner switches of chromosome cores, usually with
immunofluorescence or electron microscopy.
To what extent associations are homologous or heterologous is not obvious unless additional identifiers are used.
In the case of human, mouse, and rat meiotic chromosomes,
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Fig. 2. A demonstration of the colour-coded analysis of meiotic
prophase chromosomes. (a) The multicolour appearance of surface-spread pachytene chromosomes. (b) The SCs and associated
chromatin of the same chromosomes shown in 2a. (c) The colour
coding of the individual bivalents by the SKY-view program. Of
particular interest is the demonstration of the heterologous association between the X and Y chromosomes. The numbers correspond with those of the mitotic mouse karyotype. The length of
a medium size SC is about 10 m.

individual chromosomes have been identified by chromosome-specific probes, such as centromere- or telomerespecific probes, satellite DNA, ribosomal DNA, repeated or

Genome Vol. 44, 2001

Fig. 3. The sorted SKY karyotypes and associated SCs of two
pachytene nuclei. The average SC lengths and the standard deviations of eight such nuclei are reported in Table 1 and presented
as a histogram in Fig. 4.

unique sequences, or artificially introduced transgenes

(Heng et al. 1994). With those aids it is potentially possible
to determine chromosome-specific activities in synapsis or
recombination in normal meiosis. For a complete analysis,
however, each pair of homologous chromosomes should be
individually recognizable so that the levels of homologous
and nonhomologous synapses can be assessed.
Multicolour spectral karyotyping (SKY) has provided excellent identification of individual chromosomes and chromosome rearrangements in mitotic cells with a unique hue
for each member of the set of 23 human and 21 mouse chro 2001 NRC Canada

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0.5
0.7
0.7
0.4
0.7
0.9
1.2
1.6
0.84
0.39
0.6
1.0
0.6
0.5
1.3
1.2
1.1
2.9
1.15
0.77
1.1
1.1
1.3
1.2
1.1
1.5
1.9
2.8
1.50
0.59

Fig. 4. Average SC lengths and standard deviations of colourcoded bivalents in eight mouse spermatocyte nuclei measured in
arbitrary length units. From this relatively small sample, it would
appear the chromosomes 5 and 15 are structurally less stable
than chromosomes 3, 8, 9, and 14. This type of analysis has not
been feasible in the past as it depends on the individual identification of each chromosome.

1.4
0.7
1.4
1.3
1.5
1.8
2.0
4.0
1.76
0.98
0.9
0.8
1.0
1.7
2.0
1.6
1.9
4.2
1.76
1.09
0.8
1.5
1.4
1.2
1.3
2.0
1.8
3.1
1.64
0.69
2.2
1.2
2.7
2.4
3.2
2.0
2.6
5.1
2.68
1.14
1
8
5
6
4
7
3
2
Average
Standard
deviation

0.9
1.3
1.5
2.0
2.0
2.3
2.8
4.3
2.14
1.06

0.9
1.4
2.1
1.5
1.4
1.8
1.5
2.7
1.66
0.54

1.5
1.1
1.3
2.7
1.2
2.1
3.3
7.1
2.54
2.00

0.9
0.7
1.2
1.9
1.4
1.7
2.0
4.1
1.74
1.06

1.2
1.2
1.4
1.7
1.3
1.8
2.8
4.2
1.95
1.05

0.8
1.1
1.4
1.4
1.6
1.5
2.0
2.1
1.49
0.43

1.5
1.4
1.7
1.2
1.6
1.8
1.6
2.4
1.65
0.35

0.8
0.6
1.8
0.8
0.6
2.4
2.4
2.1
1.48
0.48

0.5
0.9
1.1
1.2
1.4
1.7
2.1
2.5
1.42
0.65

0.7
0.9
0.8
1.0
1.8
2.0
2.3
2.4
1.49
0.71

0.6
1.1
1.3
0.9
1.3
1.4
1.4
1.9
1.24
0.39

1.2
0.8
0.8
0.9
0.9
1.4
1.8
1.8
1.20
0.42

19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2

Chromosome No.

1
Nucleus

Table 1. Average SC lengths and standard deviations of colour-coded bivalents in eight mouse spermatocyte nuclei measured in arbitrary length units.

19.0
19.5
25.5
25.9
27.9
32.9
38.5
61.3

297

Sum

Heng et al.

mosomes (Liyanage et al. 1996; Schrock et al. 1996). The

system is extensively and successfully used in the detection
of chromosome abnormalities in cell lines and malignant
growths (Liyanage et al. 1996; Liu et al. 2000; Ried et al.
1998) but its application to meiotic prophase chromosomes
where it can serve numerous purposes has so far not been reported. We demonstrate that multicolour fluorescent in situ
hybridization (FISH) can provide an unprecedented refinement in meiotic chromosome analysis with an example of
the recognition of a heterologous association of the X and Y
chromosomes and with the statistics of differential chromosome compaction at meiotic prophase.
Detection of heterologies
We anticipate that the SKY identification of individual
chromosomes will permit the quantification of mispairing at
meiotic prophase. In a number of gene disruptions, chromosome synapsis is compromised (ATM/, SPO11/,
DMC1/, SCP3/) but it is not clear if the occasional SCs
are between homologous or heterologous chromosomes. The
recognition of a heterologous association by SKY analysis is
demonstrated here by the XY pair in Figs. 2 and 3. In the
authentic SKY colour in Fig. 2a and in the colour-coded image of Figs. 2c, 3a, and 3b, the juxtaposition of the two different chromatin domains is clearly defined. From this
demonstration it seem likely that the application of this
methodology can visualize the extent of homologous and
nonhomologous synapsis in cases of disturbed meiotic
prophase.
Individual chromosome characteristics
Observations on meiotic prophase chromosomes suggest
that they do not progress through prophase in perfect synchrony. For example, some chromosomes have initiated
synapsis before others, some are late in completing synapsis,
some are arranged in a bouquet, others less so, and some
lose their association with recombination proteins before
others (Moens et al. 1998). These traits are evident relative
to the average condition of all chromosomes in the same nucleus, and the question whether deviations from the average
are chromosome-specific traits or random events can be an 2001 NRC Canada

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swered only if each chromosome can be identified. Technically this has not been possible to date. It is in this respect
that the SKY procedure can bring new insight into structural
and functional aspects of meiotic prophase chromosomes
during their critical activities in synapsis, recombination,
and disjunction.
As an example, we compare the meiotic SC karyotypes of
eight pachytene nuclei (Table 1; Fig. 4). The average SC
lengths of each chromosome in Fig. 4 show that there is a
general tendency from long to short in agreement with the
mitotic assignments by the SKY analysis. However, within
each nucleus there is only a very general correspondence between the meiotic SC length of a particular chromosome and
its mitotic karyotype position (Table 1). The variation in total lengths of all SCs in a meiotic nucleus (1961 unit
lengths in Table 1) is generally attributed to developmental
stage and to preparation effects (Moses et al. 1977).
While the variation in length of a particular bivalent relative to the others within the same nucleus has been attributed
exclusively to preparation techniques, the explanation has
not been verified because of the inability to positively identify particular chromosomes. In the present case, the relative
positions of chromosomes 5 and 15 are the most variable
within the meiotic karyotype (Fig. 4). However, rather than
crediting preparation artifacts for this variation, these chromosomes may have a more flexible structure than the others.
Conversely, chromosomes 3, 4, 8, 9, and 14 have small standard deviations indicating less variable SC lengths, and possibly a more rigid chromosome structure. A more extensive
study of SCs in SKY colour-coded chromosomes may determine if the differences in the variances are fortuitous or indicative of inherent differences in chromosome structure.

Acknowledgements
This work was supported by the start-up fund for H.H.
from the Center for Molecular Medicine and Genetics, Wayne
State School of Medicine and by the Natural Sciences and
Engineering Research Council of Canada for P.M. Additional
support was provided by the Genomics/Genetics core of the
Karmanos Cancer Institute. We thank Barbara Spyropoulos
for her help in the preparation of this manuscript.

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