View larger version (95K):
[in a new window]
|
Fig. 1. Differential staining of S. cerevisiae and S. paradoxus
genomes by FISH with DNA probes from the two species. Red, S.
cerevisiae DNA; green, S. paradoxus DNA; blue,
DAPI-counterstaining of unlabelled DNA regions. (A) Cells from a mixed S.
cerevisiae-S. paradoxus culture. The nuclei are differently marked by
simultaneous hybridisation with total genomic DNA probes from the respective
species. The halos around the nuclei stem from the differential staining of
mitochondria by hybridisation with species-specific mitochondrial DNA
sequences that were contained in the probes. (B) S. cerevisiae
x S. paradoxus nuclei show a mosaic hybridisation pattern after
simultaneous FISH with total genomic DNA from the two parental species,
indicating the intermixing of the chromosome complements. (C) Differential
labelling of the nuclei of a mixed culture with a Ty1 probe from S.
cerevisiae (red) and genomic DNA from S. paradoxus (green). Only
S. cerevisiae nuclei are labelled with the Ty1 probe. (D) FISH of a
composite single sequence probe (covering most part of the left arm of
chromosome IV) from S. cerevisiae to hybrid nuclei. Each
nucleus contains a strong (arrowheads) and a weak signal (arrows),
corresponding to S. cerevisiae and S. paradoxus chromosomes
IV, respectively. This demonstrates that species-specific single
sequence probes cross-hybridise only weakly. (E) Hybrid nuclei simultaneously
hybridised with rDNA probes from the two parental species. The two rDNA tracts
are differentially stained, which indicates the high species-specificity of
the rDNA sequences. (F) Trisomic addition strain of S. cerevisiae
with an additional chromosome IV from S. paradoxus. The
S. paradoxus chromosome occupies a distinct oblong territory (arrows)
that is delineated by the S. paradoxus probe. The S.
cerevisiae probe highlights the remainder of the nucleus. (G-K) Karyogamy
and mixing of the parental genomes in hybrid S. cerevisiae x
S. paradoxus zygotes as seen by GISH. The unstained sectors of nuclei
(arrowheads) mark the sites of nucleoli where hybridisation of labelled probes
was blocked by excess unlabelled rDNA. (G) Haploid parental nuclei in a
zygote. (H) Zygote containing a diploid hybrid nucleus after karyogamy. The
two genomes are still spatially separated. (I) Incipient intermixing of
genomes in a nucleus whose shape suggests that mitosis has started. (J)
Overview of a budding zygote at lower magnification with the nucleus in
mitosis (similar stage as in I). Phase contrast picture of the entire zygote
was merged with the FISH image of the nucleus. (K) Anaphase of a zygote
nucleus with the genomes completely mixed. Bar, 2 µm (in F, for A-F); 2
µm (in K, for G-I,K).
|
