1 Institute of Botany, University of Vienna, Rennweg 14, A-1030 Vienna,
Austria
2 Max-Planck-Institute for Molecular Genetics, Ihnestraße 73, D-14195
Berlin, Germany
* Author for correspondence (e-mail: josef.loidl{at}univie.ac.at)
Accepted 24 July 2002
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Summary |
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Key words: Yeast, Hybrid, Zygote, Nuclear architecture, FISH, Interphase, Meiosis, Chromosome
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Introduction |
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When studying chromosome behaviour in hybrids, it is of great advantage if
chromosomes derived from different species can be discriminated. Total genomic
DNA from one species was used to detect introgressed chromosomes from this
species in the nuclei of another species by in situ hybridisation
(Manuelidis, 1985;
Schardin et al., 1985
), this
technique was later also applied for the differential labelling of the two
genomes in hybrids (Schwarzacher et al.,
1989
; Le et al.,
1989
) and was termed genomic in situ hybridisation (GISH)
(Schwarzacher et al., 1989
).
These studies showed the nonrandom positioning of the two parental chromosome
sets with respect to each other in interphase nuclei and on the mitotic
spindle and stated that in hybrids of several plants, parental genomes are
separated in the nuclei of differentiated tissues. Genome separation was also
found in somatic cell hybrids in animals
(Rechsteiner and Parsons,
1976
; Zelesco and
Marshall-Graves, 1988
) and plants
(Gleba et al., 1987
), in at
least some differentiated cell types of mouse (Mus musculus x
M. spretus) hybrids (Mayer et
al., 2000a
), and in nuclei formed upon fusion of human sperm with
golden hamster eggs (Brandriff et al.,
1991
). Separation of the paternal and maternal chromosome sets is
not limited to the cells of hybrid organisms or cultured hybrid cells, but was
also observed to be maintained for several cell cycles following fertilisation
in mouse embryos (Odartchenko and
Keneklis, 1973
; Mayer et al.,
2000b
).
S. cerevisiae and S. paradoxus (syn. S.
douglasii) (Naumov and Naumova,
1990) are closely related yeast species with an estimated genome
divergence of about 8-20% determined by DNA sequence comparison of certain
coding and noncoding sequences (Herbert et
al., 1988
; Adjiri et al.,
1994
; Chambers et al.,
1996
). Natural and artificial hybrids are viable, but practically
sterile (Hawthorne and Philippsen,
1994
; Hunter et al.,
1996
). Nevertheless, rare progeny must occur since natural
introgression was observed (Naumov et al.,
1997
). Here we show that nuclear DNA sequences of the sibling
species of the genus Saccharomyces are sufficiently diverse to elicit
differential labelling by fluorescence in situ hybridisation (FISH) with
genomic DNA of S. cerevisiae and S. paradoxus.
Yeasts of the genus Saccharomyces have a primarily diplontic life
cycle, but undergo meiosis and form haploid spores upon starvation. Spores of
complementary mating types (a and ) can conjugate whereby cell
fusion is directly followed by karyogamy
(Byers, 1981
). The diploid
nucleus of the zygote replicates its DNA and enters a mitosis, the result of
which is a bud, the first cell of the diploid generation (reviewed by
Marsh and Rose, 1997
).
Here we used GISH to study the behaviour and redistribution of the parental genomes in zygotes and subsequent cells of Saccharomyces cerevisiae x S. paradoxus hybrids to ask whether the phenomenon of genome separation exists in yeasts. We also investigated the pairing of the differentially labelled genomes in meiosis. Moreover, a trisomic chromosome addition strain (with one chromosome of S. paradoxus added to a diploid set of S. cerevisiae) and a substitution strain (with two chromosomes III of S. paradoxus replacing both their homoeologous chromosomes in an otherwise pure S. cerevisiae background) were used to trace individual chromosomes in interphase by GISH.
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Materials and Methods |
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Cell culture and preparation
Vegetative cells were obtained by culturing the strains in liquid YPD at
30°C. For meiotic cell preparations, cultures were grown to a density of
2x107 cells/ml in YPA. The cells were collected by
centrifugation and resuspended in 2% (w/v) potassium acetate at a density of
4x107 cells/ml. The resulting cell suspension was incubated
shaking at 30°C to induce meiosis. For obtaining zygotes, dense
suspensions of cells of opposite mating types were thoroughly mixed by
vortexing and ultrasonication, put on YPD plates and incubated for 3 hours.
Progress of mating was monitored via phase-contrast microscopy every 30
minutes until zygotes reached the desired developmental stage (normally after
3-5 hours).
Cells were collected from liquid cultures or plates and spheroplasted with
Zymolyase 100T (140 µg/ml; Seikagaku, Tokyo) or with Zymolyase 100T plus
Novozym 234 (70 µg/ml; Sigma, St Louis, MO) in 0.8 M sorbitol supplemented
with 10 mM DTT. Spheroplasting of hybrid strains worked better if cells had
been killed by washing in 1 mM NaN3, 50 mM NaF, 10 mM EDTA in 0.9%
NaCl-solution. Spheroplasting was terminated by adding 10 volumes of ice-cold
1 M sorbitol. Cells were pelleted and resuspended at a concentration of
4x108 cells/ml. This suspension was then mixed with detergent
and fixative on a slide for spreading the cells [procedure B
(Jin et al., 2000)]. For some
mitotic nuclei and zygotes we applied the detergent after fixation
[semi-spreading procedure C (Jin et al.,
2000
)]. For detailed protocols on the preparation of yeast nuclei
see Loidl et al., 1998
(Loidl et al., 1998
).
For the preparation of morphologically well-preserved zygotes amenable to
in situ hybridisation, cells were spheroplasted, fixed with formaldehyde, put
on slides, dried and postfixed with methanol and acetone as described
previously (Gotta et al.,
1996).
Genomic in situ hybridisation (GISH)
For GISH, genomic DNA was isolated from S. cerevisiae SK1 and from
the S. paradoxus N17 strain. A plasmid containing the S.
cerevisiae Ty1 retrotransposon (Boeke
et al., 1985) and a pool of 36 PCR products of mostly single copy
sequences from the left arm of chromosome IV of S.
cerevisiae (J.F., A.L. and J.L., unpublished) were also used as probes.
Finally, probes for the nucleolar organiser regions were generated. For this,
total rDNA repeats from both species were amplified using oligos
5'-GTGCATGGCCGTTCTTAGTTGG-3' and
5'-GCGCTTACTAGGAATTCCTCG-3' as primers by long-range PCR (Expand
Long Template PCR System, Roche Diagnostics, Mannheim, Germany). Probes were
labelled by nick translation with Biotin-21-dUTP (Clontech Laboratories, Palo
Alto, CA), Biotin-14-dATP (Invitrogen, Carlsbad, CA), Digoxigenin-11-dUTP,
FITC-12-dUTP (Roche Diagnostics) or Cy3-dUTP (Amersham Pharmacia, Little
Chalfont, UK) as described (Loidl et al.,
1998
).
Labelled probes were dissolved in hybridisation solution (50% formamide,
10% dextran sulfate, 2x SSC) to a final concentration of 10
ng/µl for single copy and Ty FISH probes,
30 ng/µl for genomic
probes in hybrids, and
50 ng/µl for genomic probes in substitution and
addition lines. After 5 minutes of denaturation at 95°C the probes were
dropped onto the slides, denatured for 10 minutes at 80°C and hybridised
for at least 36 hours at 37°C. For FISH with total genomic DNA, the
disproportionally strong hybridisation to rRNA gene tracts was blocked by
adding unlabelled rDNA in
10-fold excess. Post hybridisation washes were
carried out in 50% formamide in 2x SSC (37°C), 2x SSC
(37°C) and 1x SSC (room temperature) for 5 minutes each.
Subsequently, biotinylated probes were detected using FITC-conjugated avidin
(Sigma) and Digoxigenin-11-dUTP labelled probes were detected by
anti-Digoxigenin-Rhodamine (Roche Diagnostics). Finally, slides were mounted
under a coverslip in Vectashield mounting medium for fluorescence (Vector
Laboratories, Burlingame, CA) supplemented with 1 µg/ml DAPI (4'
6-diamidino-2-phenylindole) as a DNA-specific counter-stain.
Microscopy and evaluation
After FISH and detection preparations were evaluated using a Zeiss Axioskop
epifluorescence microscope equipped with single-band-pass filters for the
excitation of red, green and blue. Images of high magnification were obtained
using a cooled black and white CCD camera controlled by IPLab Spectrum
software (Scanalytics, Fairfax, VA) or the ISIS imaging system (MetaSystems,
Altlussheim, GER).
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Results |
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To investigate to what degree diverged single copy or repetitive Ty1 sequences contribute to the genome-specific hybridisation signals obtained, separate hybridisations were performed with these two genome fractions. First, a S. cerevisiae Ty1 probe was hybridised to preparations containing equal proportions of S. cerevisiae and S. paradoxus cells. Half of the nuclei exhibited a speckled FISH pattern whereas the others were virtually devoid of Ty1 signals (Fig. 1C), which indicates preferential hybridisation of the Ty1. Those nuclei that were devoid of the Ty1 signal were labelled by FISH with S. paradoxus genomic probe (Fig. 1C). This confirmed that the Ty1 probe indeed recognises S. cerevisiae nuclei. In order to test whether nonrepetitive sequences are able to elicit differential FISH signals, we used a pool of 36 PCR products of mostly single copy sequences from the left arm of S. cerevisiae chromosome IV. This compound probe produced a strong and a weak linear signal in hybrid nuclei (Fig. 1D), which demonstrates that differences in single copy sequences contribute significantly to the discrimination of the two genomes by GISH. It also shows that in the hybrid (which had been kept in culture for at least 50 generations) this 450 kb region had maintained its integrity.
Hybridisation with genomic DNAs was particularly strong at rDNA regions where the signal outshone the remainder of the nucleus. To reveal the signals at the nuclear DNA, rDNA hybridisation was suppressed by addition of an excess of unlabelled rDNA to the hybridisation probe. Species-specific rDNA probes produced differential staining of the two NORs in diploid hybrid nuclei (Fig. 1E). Thus, rDNA repeats seem to have undergone a similar degree of divergence between the two species, as did the genome as a whole.
The genomic DNA extracted from the two species also contained mitochondrial DNA. In semi-spread preparations of cells, nuclei were surrounded by DAPI-bright mitochondria that upon GISH showed the same species-specific labelling as the corresponding nuclei (Fig. 1A).
The relative positioning of the two genomes in hybrid nuclei
GISH on S. cerevisiae x S. paradoxus hybrids showed
that the parental chromosome complements are intermingled, with hybrid nuclei
containing a mosaic of red and green patches
(Fig. 1B). In the 30 well
preserved nuclei that were scored, none of the parental chromatin sets formed
a single contiguity. This contrasts with reports on the separation of parental
genomes in the nuclei of hybrid plants and somatic cell hybrids. Since zygotic
genome separation was reported from mammalian embryos, we wanted to see
whether a newly formed hybrid nucleus would maintain parental genome
separation for some time. We mated S. cerevisiae and S.
paradoxus cells of opposite mating types on plates and prepared zygotes
at different stages of karyogamy for GISH. In two-nucleate zygotes the
parental nuclei could be well differentiated
(Fig. 1G). Immediately after
karyogamy the two genomes were separate and the nucleoli were situated at the
distal ends of the elongated nucleus (Fig.
1H). The position of the nucleoli suggests that the two chromosome
sets are facing each other with their centromeres since the nucleolus and
centromeres occupy opposite poles of nuclei
(Jin et al., 2000). Moreover,
during karyogamy nuclei fuse at the regions of the spindle pole bodies (SPBs)
(Marsh and Rose, 1997
) to
which the centromeres are attached (Jin et
al., 2000
). Fused elongated zygote nuclei that had their
longitudinal axis oriented transversal to the long axis of the zygote showed
the beginnings of intermixing of the parental chromosome sets
(Fig. 1I). FISH on zygotes with
well-preserved cell walls confirmed that these oblong nuclei represent early
mitotic nuclei that just enter or pass through the neck of the zygotic bud
(Fig. 1J). This suggests that
intermixing starts with the onset of zygotic mitosis. By the end of the
zygotic mitosis, the mingling of chromosomes was virtually complete
(Fig. 1K).
Delineation of individual chromosomes in addition and substitution
strains
GISH on nuclei of addition strains or substitution strains was applied to
delineate entire chromosomes. To this end we used or constructed S.
cerevisiae strains in which an additional chromosome IV from
S. paradoxus was present (strain SKC5) or where both authentic
chromosomes III were replaced by their S. paradoxus
homoeologs (strain SLY2007). Hybridisation of these strains with S.
paradoxus genomic DNA clearly delineated the S. paradoxus
chromosomes in many of the interphase nuclei
(Fig. 1F). This indicates a
territorial organisation of yeast chromosomes in at least some stages of
interphase.
Observations in meiosis
It was reported that meiotic recombination between an S.
cerevisiae and S. paradoxus homeologous chromosome III
pair is decreased by 25-fold compared with homologs in S. cerevisiae
(Chambers et al., 1996) and
that spore viability in the complete hybrid is only 1%
(Hunter et al., 1996
). We
therefore wanted to study whether and how the homoeologous genomes pair in
hybrid meiosis. Since the strain in which the genetical studies had been
performed turned out to be unfavourable for meiotic cytology, we carried out
our investigation in the hybrid strain SLY2006 with SK1 as the S.
cerevisiae parent. In this strain spore viability was 7% (10 of 144). We
prepared whole mount spreads of synaptonemal complexes (SCs) and investigated
them by immunostaining of the SC component Zip1
(Sym et al., 1993
), by
electron microscopy and by GISH. Zip1 is a part of the transversal filaments
and it is present between (homologously and nonhomologously) synapsed regions
of chromosomes at zygotene and pachytene of meiosis
(Sym et al., 1993
).
Immunostaining of hybrid nuclei showed several long individual threads of Zip1
indicating extensive synapsis (Fig.
2A). Electron microscopy of silver-stained synaptonemal complexes
produced a more complex image, since it reveals not only the synapsed
chromosome regions but also unpaired axial elements at pairing partner
switches. It was found that, unlike in non-hybrid pachytenes, the axes of many
chromosomes engaged in synapsis with changing partners
(Fig. 2B). This promiscuous
behaviour was also observed in pachytene SCs of the hybrid strain NHD47 that
had been studied by Hunter et al. (Hunter
et al., 1996
). There are two possible explanations for the
switching of synaptic partners. First, chromosomes of the two species might
not be co-linear (i.e. regions homologous to a single chromosome in one
species are dispersed over several chromosomes in the other), so that
chromosomes have to switch partners in order to achieve homologous synapsis.
This possibility is considered unlikely (see Discussion). Alternatively,
synapsis could occur between nonhomologous regions. GISH on pachytene nuclei
showed that not only green and red genome portions were associated
(Fig. 2C), but occasionally
there were two chromosome regions of the same color paired
(Fig. 2D). This indicates that
nonhomologous pairing of chromosomes or chromosome regions within the same
species does occur. It further suggests that the pairing of S.
cerevisiae and S. paradoxus chromosomes may also be at least
partially heterologous.
|
In pachytene nuclei of chromosome addition and substitution strains, chromosomes derived from S. paradoxus could be delineated. In contrast to the hybrid, meiotic pairing was undisturbed and the synapsis of S. paradoxus chromosomes III in the S. cerevisiae background was normal (Fig. 2E).
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Discussion |
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Pulsed field-gel electrophoresis showed that chromosome number and sizes
are largely conserved between the two species
(Naumov et al., 1992;
Hunter et al., 1996
).
Moreover, all of 15 genes investigated located to the corresponding
chromosomes in the two species (Naumov et
al., 1992
; Hunter et al.,
1996
). Likewise, Southern hybridisation on electrophoretic
karyotypes with probes from close to the centromere and one from near each end
of each of the 16 chromosomes demonstrated that the S. paradoxus
karyotype is colinear (i.e. they show no detectable translocations) with that
of S. cerevisiae (Fischer et al.,
2000
). The fact that S. cerevisiae strains in which the
authentic chromosome III is replaced by the corresponding S.
paradoxus chromosome grow vigorously under laboratory conditions
[(Chambers et al., 1996
) and
this study], demonstrates that the genes on this chromosome can substitute for
the roughly 200 genes on S. cerevisiae chromosome III, and
confirms the colinearity (synteny) of the chromosomes between the two species.
However, recombination between the homoeologous genomes is low, as was shown
by the high frequencies of aneuploidy and low frequencies of genetic exchange
of the rare offspring of hybrids (Hunter
et al., 1996
).
Yeast hybrid nuclei do not show separation of parental genomes
We co-cultivated haploid S. cerevisiae and S. paradoxus
strains of opposite mating types to obtain hybrid zygotes. In preparations
from these cultures, we found that even the earliest diploid nuclei (except
those in the zygotes themselves) had the two genomes arranged at random. This
indicates that their intermixing starts during or soon after karyogamy; either
in the zygote or during the first mitosis. Hence also the nuclei of S.
cerevisiae x S. paradoxus hybrids that had been in culture
for extended periods contained entirely intermixed parental chromosome sets.
The rapid randomisation of chromosome distribution is in contrast to the
separation of parental genomes that has been observed in a wide range of
hybrid cell types and organisms. Genome separation was described in several
hybrid plants and in cultured hybrid cells (see Introduction).
Since the different genomes in yeast hybrids appear to mix during or
immediately after karyogamy, it is reasonable to assume that the chromosomes
in normal (non-hybrid) matings will also intermingle. This is different from
fertilisation in animals, where it was shown that the parental genomes remain
spatially separated for several cell generations following the zygote
(Odartchenko and Keneklis,
1973; Mayer et al.,
2000c
). Sperm chromatin undergoes extensive remodelling and
modification that causes its transition from the densely packed and
transcriptionally inactive state in the sperm to an open chromatin
configuration in the male pronucleus (e.g.
Brandriff et al., 1991
). This
remodelling is accompanied by rapid DNA demethylation
(Mayer et al., 2000b
) and
probably other epigenetic modifications, which render the parental genomes in
the zygote structurally and transcriptionally different
(Vielle-Calzada et al., 2000
).
Spatial separation may help to maintain these differences. Continued
inactivation of one parental genome could be of functional significance since
silencing of one genome will extend a functionally haploid state in diplontic
or diplohaplontic organisms, in which the haplophase is the only period when a
defective recessive allele can be efficiently selected against
(Vielle-Calzada et al., 2000
).
However, this requirement does not apply to yeast, as this organism normally
forms several haploid cell generations after sporulation.
Yeast chromosomes occupy distinct territories in interphase
In metazoans and plants individual chromosomes occupy well-separated
regions of the interphase nucleus. This territorial organisation of
chromosomes has been proposed to be important for the functional
compartmentalisation of the cell nucleus
(Cremer et al., 1993). In
yeast, the occurrence of ectopic mitotic recombination events between loci
within or between chromosomes at similar frequencies has led to the
interpretation that this organism lacks chromosome territories
(Haber and Leung, 1996
). The
observation that ectopic recombination is efficient suggests that chromatin
fibers are loosely packaged and intermix with chromatin of other chromosomes.
However, by using GISH, we observed dense, mutually exclusive stained areas in
hybrid nuclei (Fig. 1B) and
distinct domains for individual chromosomes in addition and substitution
strains (Fig. 1F). This
provides evidence that in interphase nuclei of budding yeast there exist
chromosome territories similar to those in higher eukaryotes.
Meiotic pairing in the hybrid is partially random
In meiosis of the hybrid, chromosomes do not pair as bivalents but they are
engaged in pairing partner switching that produces multivalents
(Fig. 2B). Since the karyotypes
of the two species do not seem to differ by multiple translocations (see
above) the synaptic switches do not reflect pairing of homologous regions
dispersed over different chromosomes. These switches are rather due to
nonhomologous synapsis, as is, for instance, also found in the meiosis of
haploid yeast, where chromosomes lack homologous partners
(Loidl et al., 1991). The
occurrence of nonhomologous synapsis is also supported by the observation of
occasional pairing between chromosomes of one and the same parental set
(Fig. 2D).
In the budding yeast, SC formation depends on the initiation of
recombination (Alani et al.,
1990; Padmore et al.,
1991
) and seems to initiate at sites where recombination has
occurred (Agarwal and Roeder,
2000
). In the absence of homologous chromosomes recombination
tends to occur between minor ectopic homologies and promotes the formation of
nonhomologous SC (Loidl and Nairz,
1997
). Since in the S. cerevisiae x S.
paradoxus hybrids there is considerable pairing of nonhomologous
chromosomes, it appears that sequence homology between the parental genomes is
not sufficient to support exclusive homoeologous recombination and synapsis.
Thus one could speculate that homoeologous recombination is rarely initiated
and/or it does not progress to a stage that promotes homoeologous synapsis.
While the NHD47 hybrid produces only 1.2% viable spores in which genetic
exchange is reduced and aneuploidy is high, bybrids lacking the mismatch
repair genes PMS1 or MSH2 are improved with respect to spore
viability and recombination and segregation
(Hunter et al., 1996
).
Chambers et al. (Chambers et al.,
1996
) and Hunter et al.
(Hunter et al., 1996
) proposed
that recombination that initiates between regions of inadequate homology (e.g.
the homoeologous chromosomes of the hybrid) is abolished by the mismatch
repair system. It will be interesting to test whether the increase in
homoeologous crossing over after disruption of PMS1 or MSH2
is accompanied by more extensive homoeologous synapsis.
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Acknowledgments |
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