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HARRY SCHERTHAN and EDGAR TRELLES-STICKEN
phase B. Despite its powerful genetics, S. cerevisiae cytology is further
hampered by the very small size of the object: diploid nuclei are only
2-4 Jlm in diameter and harbor 32 chromosomes with a total DNA content
of24 Mbp of predominantly unique DNA sequences (Goffeau et al. 1997).
For these reasons, light and electron microscopic studies of yeast metaphase chromosomes at best revealed tiny chromatin lumps which are
hardly reminiscent of the metaphase chromosomes of more complex eukaryotes (Kater 1927, Wintersberger et al. 1975). Unlike in mitosis, pachytene chromosome spreads (bivalents) can be obtained from meiotic yeast
cultures when homologues are connected by their joint synaptonemal
complex (SC; Kuroiwa et al. 1984, Dresser and Giroux 1988).
SCs can be visualized in intact nuclei by 3D electron microscopy or by
silver staining in surface spread meiocyte nuclei (for details see Esposito
et al. 1990, Loidl et al. 1998). Introduction of FISH to yeast cytology
(Scherthan et al. 1992, Uzawa et al. 1992) provided the first means to delineate individual chromosomes and their subregions in spreads and undisrupted nuclei. Therefore, molecular yeast cytology has become a major
tool to physically monitor chromosome behavior during wild-type and
mutant mitotic and meiotic cell cycles (Funabiki et al. 1993, Loidl et al.
1994, Nag et al. 1995, Gotta et al. 1996, Guacci et al. 1997, Trelles-Sticken
et al. 1999). Below, we outline a protocol for yeast FISH which is routinely
used in our lab.
Materials
- Standard equipment used in the cytological laboratory including: Coplin jars with lids, glass slides, coverslips, micropipettes, heating block,
plastic or metal boxes, microcentrifuge and incubator.
- Phase contrast and fluorescence microscope equipped with appropriate filter sets for excitation of the different f1uorochromes applied. A
conventional or preferably a digital image recording and processing
system.
Reagents - Antifade solution: Vectashield (Vector Labs) is recommended as
mounting medium for fluorescence microscopy, since it efficiently reduces fading of f1uorochromes during microscopic analysis (Florijn et
al. 1995)
- Avidin-FITC (e.g., ExtrAvidin-f1uorescein; Sigma)
HARRY SCHERTHAN and EDGAR TRELLES-STICKEN
phase B. Despite its powerful genetics, S. cerevisiae cytology is further
hampered by the very small size of the object: diploid nuclei are only
2-4 Jlm in diameter and harbor 32 chromosomes with a total DNA content
of24 Mbp of predominantly unique DNA sequences (Goffeau et al. 1997).
For these reasons, light and electron microscopic studies of yeast metaphase chromosomes at best revealed tiny chromatin lumps which are
hardly reminiscent of the metaphase chromosomes of more complex eukaryotes (Kater 1927, Wintersberger et al. 1975). Unlike in mitosis, pachytene chromosome spreads (bivalents) can be obtained from meiotic yeast
cultures when homologues are connected by their joint synaptonemal
complex (SC; Kuroiwa et al. 1984, Dresser and Giroux 1988).
SCs can be visualized in intact nuclei by 3D electron microscopy or by
silver staining in surface spread meiocyte nuclei (for details see Esposito
et al. 1990, Loidl et al. 1998). Introduction of FISH to yeast cytology
(Scherthan et al. 1992, Uzawa et al. 1992) provided the first means to delineate individual chromosomes and their subregions in spreads and undisrupted nuclei. Therefore, molecular yeast cytology has become a major
tool to physically monitor chromosome behavior during wild-type and
mutant mitotic and meiotic cell cycles (Funabiki et al. 1993, Loidl et al.
1994, Nag et al. 1995, Gotta et al. 1996, Guacci et al. 1997, Trelles-Sticken
et al. 1999). Below, we outline a protocol for yeast FISH which is routinely
used in our lab.
Materials
- Standard equipment used in the cytological laboratory including: Coplin jars with lids, glass slides, coverslips, micropipettes, heating block,
plastic or metal boxes, microcentrifuge and incubator.
- Phase contrast and fluorescence microscope equipped with appropriate filter sets for excitation of the different f1uorochromes applied. A
conventional or preferably a digital image recording and processing
system.
Reagents - Antifade solution: Vectashield (Vector Labs) is recommended as
mounting medium for fluorescence microscopy, since it efficiently reduces fading of f1uorochromes during microscopic analysis (Florijn et
al. 1995)
- Avidin-FITC (e.g., ExtrAvidin-f1uorescein; Sigma)