27 Yeast FISH: Delineation of Chromosomal Targets in Vegetative and Meiotic Yeast Cells
343
(Scherthan et al. 1993, 1994, Guacci et al. 1994,1997). Proteases can be
used to effectively remove cytoplasm and nuclear proteins, but in our
hands proteolytic treatment, like harsh detergent treatment, resulted in
detrimental effects on yeast nuclear structure. We found that repeated
denaturation can be used as an alternative to proteolytic pre-treatment.
- Composite DNA probes for the delineation of whole chromosomes or
chromosomal subregions like telomeres or centromeres may be produced by combining several chromosome specific phage or plasmid
clones (Scherthan et al. 1992, Gotta et al. 1996, Jin et al. 1998). Note
that probe DNAs which are situated in duplicated regions of the yeast
genome (Goffeau et al. 1997) or contain t-RNA genes or TY elements,
may cause supernumerary signals which generally impair interpretation of the FISH results.
- The protocols given above work in our hands on vegetative and meiotic
cells and may be combined with immunostaining of nuclear proteins
by performing FISH detection and immunodecoration of nuclear antigens simultaneously (Trelles-Sticken et al. 1999). Other protocols
which use coagulating fixatives instead of cross linking with formaldehyde are available (Gotta et al. 1999). Hence, protocols which use
different fixation regimens should be tested and compared for optimal
performance in a particular application.
- Images may be recorded with conventional microphotography, digital
image acquisition systems like cooled CCD cameras or laser scanning
microscopes. Deconvolution may be used to reduce noise in the gray
scale images.
Acknowledgements. We would like to thank Drs J. Bahler, B. Dujon, D. Kaback, J. Loidl and
E. Louis for help with the materials and J. Loidl for an introduction to the spreading technique. This work was supported by a grant of the Deutsche Forschungsgemeinschaft
(SCH350/8-3).
References
Dresser ME, Giroux CN (1988) Meiotic chromosome behavior in spread preparations
of yeast. J Cell Bioi 106:567-573.
Esposito RS, Dresser ME, Breitenbach M (1990) Identifying sporulation genes, Visualizing Synaptonemal Complexes, and Large Scale Spore and Spore Wall Purification.
Guide to Yeast Genetics and Molecular Biology. 194:110-131.
343
(Scherthan et al. 1993, 1994, Guacci et al. 1994,1997). Proteases can be
used to effectively remove cytoplasm and nuclear proteins, but in our
hands proteolytic treatment, like harsh detergent treatment, resulted in
detrimental effects on yeast nuclear structure. We found that repeated
denaturation can be used as an alternative to proteolytic pre-treatment.
- Composite DNA probes for the delineation of whole chromosomes or
chromosomal subregions like telomeres or centromeres may be produced by combining several chromosome specific phage or plasmid
clones (Scherthan et al. 1992, Gotta et al. 1996, Jin et al. 1998). Note
that probe DNAs which are situated in duplicated regions of the yeast
genome (Goffeau et al. 1997) or contain t-RNA genes or TY elements,
may cause supernumerary signals which generally impair interpretation of the FISH results.
- The protocols given above work in our hands on vegetative and meiotic
cells and may be combined with immunostaining of nuclear proteins
by performing FISH detection and immunodecoration of nuclear antigens simultaneously (Trelles-Sticken et al. 1999). Other protocols
which use coagulating fixatives instead of cross linking with formaldehyde are available (Gotta et al. 1999). Hence, protocols which use
different fixation regimens should be tested and compared for optimal
performance in a particular application.
- Images may be recorded with conventional microphotography, digital
image acquisition systems like cooled CCD cameras or laser scanning
microscopes. Deconvolution may be used to reduce noise in the gray
scale images.
Acknowledgements. We would like to thank Drs J. Bahler, B. Dujon, D. Kaback, J. Loidl and
E. Louis for help with the materials and J. Loidl for an introduction to the spreading technique. This work was supported by a grant of the Deutsche Forschungsgemeinschaft
(SCH350/8-3).
References
Dresser ME, Giroux CN (1988) Meiotic chromosome behavior in spread preparations
of yeast. J Cell Bioi 106:567-573.
Esposito RS, Dresser ME, Breitenbach M (1990) Identifying sporulation genes, Visualizing Synaptonemal Complexes, and Large Scale Spore and Spore Wall Purification.
Guide to Yeast Genetics and Molecular Biology. 194:110-131.