SpringerProtocols: Abstract: 10. Whole-Mount Fluorescence In Situ Hybridization to Chromosomes of Embryos

10. Whole-Mount Fluorescence In Situ Hybridization to Chromosomes of Embryos

By: Daryl S. Henderson²

Abstract

Full Text

Download PDF (760K)

Whole-mount fluorescence in situ hybridization (FISH) to chromosomes of Drosophila embryos is used to pinpoint the position of a chromosomal segment of interest, specified by the DNA probe(s), within a “preserved” three-dimensional nuclear structure. This technique has been used to (1) visualize the relative positions of homologous loci during the onset of somatic chromosome pairing (1,2), (2) establish whether separation of sister chromatids had occurred in mutants arrested at the metaphase/anaphase transition (3–5), (3) karyotype zygotes (6), and (4) ascertain the ploidy of nuclei of a checkpoint mutant (7). The basic embryo-FISH technique can be combined with antibody staining to permit simultaneous detection of both a hybridized DNA probe and a specific subcellular structure, such as the nuclear envelope, permitting spatial relationships to be defined (8,9). Protocols for both techniques are included in this chapter.

Affiliation(s): (2) Department of Pharmacological Sciences, State University of New York at Stony Brook, NY

Book Title: Drosophila Cytogenetics Protocols

Series: Methods in Molecular Biology | Volume: 247 | Pub. Date: Nov-04-2003 | Page Range: 235-247 | DOI: 10.1385/1-59259-665-7:235

Subject: Genetics/Genomics

References

Download References

1.	Hiraoka, Y., Dernburg, A. F., Parmelee, S. J., Rykowski, M. C., Agard, D. A., and Sedat, J. W. (1993) The onset of homologous chromosome pairing during Drosophila melanogaster embryogenesis. J. Cell Biol. 120, 591–600.

2.	Gemkow, M. J., Verveer, P. J., and Arndt-Jovin, D. J. (1998) Homologous association of the Bithorax-Complex during embryogenesis: consequences for transvection in Drosophila melanogaster. Development 125, 4541–4552.

3.	Sigrist, S., Jacobs, H., Stratmann, R., and Lehner, C. F. (1995) Exit from mitosis is regulated by Drosophila fizzy and the sequential destruction of cyclins A, B and B3. EMBO J. 14, 4827–4838.

4.	Stratmann, R. and Lehner, C. F. (1996) Separation of sister chromatids in mitosis requires the Drosophila pimples product, a protein degraded after the metaphase/anaphase transition. Cell 84, 25–35.

5.	Bhat, M., Philp, A. V., Glover, D. M., and Bellen, H. J. (1996) Chromatid segregation at anaphase requires the barren product, a novel chromosome-associated protein that interacts with topoisomerase II. Cell 87, 1103–1114.

6.	Dernburg, A. F., Daily, D. R., Yook, K. J., Corbin, J. A., Sedat, J. W., and Sullivan, W. (1996) Selective loss of sperm bearing a compound chromosome in the Drosophila female. Genetics 143, 1629–1642.

7.	Forgarty, P., Campbell, S. D., Abu-Shumays, R., et al. (1997) The Drosophila grapes gene is related to checkpoint gene chk1/rad27 and is required for late syncitial division fidelity. Curr. Biol. 7, 418–426.

8.	Gemkow, M. J., Buchenau, P., and Arndt-Jovin, D. J. (1996) FISH in whole-mount Drosophila embryos. RNA: activation of a transcriptional locus, DNA: gene architecture and expression. Bioimaging 4, 107–120.

9.	Marshall, W. F., Dernburg, A. F, Harmon, B., Agard, D. A., and Sedat, J. W. (1996) Specific interactions of chromatin with the nuclear envelope: positional determination within the nucleus in Drosophila melanogaster. Mol. Biol. Cell 7, 825–842.

10.	Lohe, A. R., Hilliker, A. J., and Roberts, P. A. (1993) Mapping simple repeated DNA sequences in heterochromatin of Drosophila melanogaster. Genetics 134, 1149–1174.

11.	Bonaccorsi, S. and Lohe, A. (1991) Fine mapping of satellite DNA sequences along the Y chromosome of Drosophila melanogaster: relationships between satellite sequences and fertility factors. Genetics 129, 177–189.

12.	Csink, A. K. and Henikoff, S. (1998) Large-scale chromosomal movements during interphase progression in Drosophila. J. Cell Biol. 143, 13–22.

13.	Carmena, M., Abad, J. P., Villasante, A., and Gonzalez, C. (1993) The Drosophila melanogaster dodecasatellite sequence is closely linked to the centromere and can form connections between sister chromatids during mitosis. J. Cell Sci. 105, 41–50.

14.	Dernburg, A. F., Broman, K. W., Fung, J. C., et al. (1996) Perturbation of nuclear architecture by long-distance chromosome interactions. Cell 85, 745–759.

15.	Gunawardena, S. and Rykowski, M. (1994) Looking at diploid interphase chromosomes. Methods Cell Biol. 44, 393–409.

16.	Gunawardena, S., Heddle, E., and Rykowski, M. C. (1995) “Chromosome puffing” in diploid nuclei of Drosophila melanogaster. J. Cell Sci. 108, 1863–1872.

17.	Foe, V. E. and Alberts, B. M. (1985) Reversible chromosome condensation induced in Drosophila embryos by anoxia: visualization of interphase nuclear organization. J. Cell Biol. 100, 1623–1636.

18.	Marshall, W. F., Fung, J. C., and Sedat, J. W. (1997) Deconstructing the nucleus: global architecture from local interactions. Curr. Opin. Genet. Dev. 7, 259–263.

19.	Feinberg, A. P. and Vogelstein, B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6–13.

20.	Feinberg, A. P. and Vogelstein, B. (1984) “A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.” Addendum. Anal. Biochem. 137, 266–267.

21.	Rigby, P. W. J., Dieckmann, M., Rhodes, C., and Berg, P. (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113, 237–251.

22.	Schmitz, G. G., Walter, T., Seibl, R., and Kessler, C. (1991) Nonradioactive labeling of oligonucleotides in vitro with the hapten digoxigenin by tailing with terminal transferase. Anal. Biochem. 192, 222–231.

23.	Pathak, S., Choi, S.-K., Arnheim, N., and Thompson, M. E. (2001) Hydroxylated quantum dots as probes for in situ hybridization. Am. J. Chem. Soc. 123, 4103–4104.

24.	Goldman, E. R., Balighian, E. D., Mattoussi, H., et al. (2002) Avidin: a natural bridge for quantum dot-antibody conjugates. Am. J. Chem. Soc. 124, 6378–6382.

25.	Stuurman, N., Maus, N., and Fisher, P. A. (1995) Interphase phosphorylation of the Drosophila nuclear lamin: site-mapping using a monoclonal antibody. J. Cell Sci. 108, 3137–3144.

26.	Augustin, M. A., Ankenbauer, W., and Angerer, B. (2001) Progress toward single-molecule sequencing: enzymatic synthesis of nucleotide-specifically labeled DNA. J. Biotechnology 86, 289–301.

27.	Hengen, P. N. (1996) Carriers for precipitating nucleic acids. Trends Biochem. Sci. 21, 224–225.

28.	Leversha, M. A. (2001) Mapping of genomic clones by fluorescence in situ hybridization. Methods Mol. Biol. 175, 109–127.

29.	Watters, A. D., Stacey, M. W., and Bartlett, S, J. M. (2002) A modified nick translation method used with FISH that produces reliable results with archival tissue sections. Mol. Biotechnology 20, 257–260.

30.	Dernburg, A. (1998) Whole-mount fluorescence in situ hybridization to Drosophila chromosomal DNA. In Cells: A Laboratory Manual, Vol. 3., Subcellular Localization of Genes and Their Products (Spector, D. L., Goldman, R. D., and Leinwand, L. A., eds.), Cold Spring Harbor Laboratory Press, Plainview, NY, pp. 115.1–115.18.

31.	Dernburg, A. F. and Sedat, J. W. (1998) Mapping three-dimensional chromosome architecture in situ. Methods Cell Biol. 53, 187–233.

32.	Spathas, D. H. and Ferguson-Smith, M. A. (1993) A simplified one-step procedure for enhanced detection of biotinylated probes with fluorescein conjugates. Trends Genet. 9, 262.

33.	Raap, A. K., Vandecorput, M., Vervenne, R., Van Gijlswijk, R., Tanke, H. J., and Wiegant, J. (1995) Ultra-sensitive FISH using peroxidase-mediated deposition of biotin-or fluorochrome tyramides. Hum. Mol. Genet. 4, 529–534.

34.	Van Gijlswijk, R., Wiegant, J., Raap, A. K., and Tank, J. H. J., (1996) Improved localization of fluorescent tyramides for fluorescence in situ hybridization using dextran sulfate and polyvinyl alcohol. Histochem, J. Cytochem. 44, 389–392.

Comments (Loading...)

Post comment/View all comments