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HARRY SCHERTHAN and EDGAR TRELLES-STICKEN
quence of the yeast genome (http://genome-www.stanford.edu/Saccharomyces) primers covering the region of interest may be designed and long
range peR can be used to produce potent FISH probes. Generally, DNA
probes larger than 6 kb and probe concentrations of 20-50 ng/1l1 are required to generate distinctly visible hybridization signals (Scherthan et al.
1992, 1994). It is recommended to determine the size and amount of labeled probe by running an aliquot e1 20 ) of the nick translation reaction in
a 1% agarose mini gel.
DNA-Probes are preferably labeled with haptens like biotin, digoxigenin or estradiol. Haptens are detected with appropriate fluorochrome-conjugated antibodies which yield fluorescent signals which are readily seen
upon visual inspection (see subprotocol 3.). The two color FISH scheme
presented below (Fig. 1) also allows three colors to be simultaneously observed under the microscope, where the third color is induced by 1:1 ratio
mixing of biotin and digoxigenin labels (Fig. lc, h). We also used direct
fluorochrome labeling but this usually results in weak hybridization sigFig. 1. FISH on spread and undisrupted S. cerevisiae nuclei. a Two-color chromosome
painting reveals paired and separated chromosomes I (red, rhodamine) and III (green,
fluorescein) in spread meiocyte nuclei (DAPI, blue) obtained 2 h after induction of sporulation. Conventional fluorescence microscopy. Bars in a-f represent 5 !Jm. b Spread pachytene nucleus showing delineated chromosome I (red) and III (green) bivalents. Conventional fluorescence microscopy. c Multi-color FISH delineates bivalents of chromosomes
I (red), III (green) and V (orange) in an extensively spread pachytene nucleus (DAPI,
blue) (conventional fluorescence microscopy). d FISH with a composite probe for all telomeres (green) and centromeres (red) to cells obtained during mitotic growth shows the
presence of a distinct centromere cluster and peripheral telomere aggregates in (i) a structurally preserved nucleus, (ii) a mildly spread nucleus, and in (iii) an extensively spread
nucleus. Note that telomere signals disperse upon enhanced spreading, while centromeres
don't. (e) Undisrupted and spread meiocyte at the bouquet stage. Telomeres (green) form a
single cluster, while centromeres (red) are dispersed. This is more obvious in the larger
spread meiocyte to the right. f Telomere (green) and cosmid FISH (red, tagging a chr.
Xl region) to undisrupted nuclei. Telomere clusters are peripherally distributed in a vegetative nucleus with separated cosmid signals (left). The meiocyte nucleus to the right displays paired cosmids (one signal) and a single telomere cluster (green; bouquet stage).
g-i FISH to spreads of diploid Schizosaccharomyces pombe cells (note that wild-type
S. pombe is haploid, n=3). g Metaphase figure with differentially marked telomeres of chromosomes 2 by FISH with two cosmid probes (red, green). h Horse tail nucleus obtained at
meiotic prophase (see Kohli 1994). Telomeres (red) and adjacent rONA (orange, by redl
green labeling) mark the leading edge of this bouquet nucleus, while centromeres (green)
locate near the opposite end. i Painting of chromosomes 1 (green) and 2 (red) in a mildly
spread diploid nucleus. Homologue association leads to one signal which delineates a joint
territory of each chromosome pair. Bar in i: 2 !Jm, it also applies to g and h
HARRY SCHERTHAN and EDGAR TRELLES-STICKEN
quence of the yeast genome (http://genome-www.stanford.edu/Saccharomyces) primers covering the region of interest may be designed and long
range peR can be used to produce potent FISH probes. Generally, DNA
probes larger than 6 kb and probe concentrations of 20-50 ng/1l1 are required to generate distinctly visible hybridization signals (Scherthan et al.
1992, 1994). It is recommended to determine the size and amount of labeled probe by running an aliquot e1 20 ) of the nick translation reaction in
a 1% agarose mini gel.
DNA-Probes are preferably labeled with haptens like biotin, digoxigenin or estradiol. Haptens are detected with appropriate fluorochrome-conjugated antibodies which yield fluorescent signals which are readily seen
upon visual inspection (see subprotocol 3.). The two color FISH scheme
presented below (Fig. 1) also allows three colors to be simultaneously observed under the microscope, where the third color is induced by 1:1 ratio
mixing of biotin and digoxigenin labels (Fig. lc, h). We also used direct
fluorochrome labeling but this usually results in weak hybridization sigFig. 1. FISH on spread and undisrupted S. cerevisiae nuclei. a Two-color chromosome
painting reveals paired and separated chromosomes I (red, rhodamine) and III (green,
fluorescein) in spread meiocyte nuclei (DAPI, blue) obtained 2 h after induction of sporulation. Conventional fluorescence microscopy. Bars in a-f represent 5 !Jm. b Spread pachytene nucleus showing delineated chromosome I (red) and III (green) bivalents. Conventional fluorescence microscopy. c Multi-color FISH delineates bivalents of chromosomes
I (red), III (green) and V (orange) in an extensively spread pachytene nucleus (DAPI,
blue) (conventional fluorescence microscopy). d FISH with a composite probe for all telomeres (green) and centromeres (red) to cells obtained during mitotic growth shows the
presence of a distinct centromere cluster and peripheral telomere aggregates in (i) a structurally preserved nucleus, (ii) a mildly spread nucleus, and in (iii) an extensively spread
nucleus. Note that telomere signals disperse upon enhanced spreading, while centromeres
don't. (e) Undisrupted and spread meiocyte at the bouquet stage. Telomeres (green) form a
single cluster, while centromeres (red) are dispersed. This is more obvious in the larger
spread meiocyte to the right. f Telomere (green) and cosmid FISH (red, tagging a chr.
Xl region) to undisrupted nuclei. Telomere clusters are peripherally distributed in a vegetative nucleus with separated cosmid signals (left). The meiocyte nucleus to the right displays paired cosmids (one signal) and a single telomere cluster (green; bouquet stage).
g-i FISH to spreads of diploid Schizosaccharomyces pombe cells (note that wild-type
S. pombe is haploid, n=3). g Metaphase figure with differentially marked telomeres of chromosomes 2 by FISH with two cosmid probes (red, green). h Horse tail nucleus obtained at
meiotic prophase (see Kohli 1994). Telomeres (red) and adjacent rONA (orange, by redl
green labeling) mark the leading edge of this bouquet nucleus, while centromeres (green)
locate near the opposite end. i Painting of chromosomes 1 (green) and 2 (red) in a mildly
spread diploid nucleus. Homologue association leads to one signal which delineates a joint
territory of each chromosome pair. Bar in i: 2 !Jm, it also applies to g and h