Summary
A restriction enzyme-nick translation procedure has been developed for localizing sites of restriction endonuclease action on chromosomes. This method involves digestion of fixed chromosome preparations with a restriction enzyme, nick translation with DNA polymerase I in the presence of biotinylated-dUTP, detection of the incorporated biotin label with streptavidinalkaline phosphatase, and finally staining for alkaline phosphatase. Results obtained on human chromosomes using a wide variety of restriction enzymes are described, and compared with results of Giemsa and Feulgen staining after restriction enzyme digestion. Results of nick translation are not in general the opposite of those obtained with Giemsa staining, as might have been expected. Although the nick translation procedure is believed to give a more accurate picture of the distribution of restriction enzyme recognition sites on chromosomes than Giemsa staining, it is clear that the results of the nick translation experiments are affected by accessibility to the enzymes of the chromosomal DNA, as well as by the extractability of the DNA.
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References
Adolph, S. (1988).In situ nick translation distinguishes between C-band positive regions on mouse chromosomes.Chromosoma 96, 102–6.
Antequera, F., Macleod, D. &Bird, A. P. (1989). Specific protection of methylated CpGs in mammalian cell nuclei.Cell 58, 507–17.
Beçak, M. L., Dos Santos, R. De C. S., Soares-Scott, M. D., Batistic, R. F. &Costa, H. (1988). Chromosome structure in man and Amphibia-Anura, restriction enzymes.Rev. Brasil. Genet. 11, 939–48.
Bianchi, M. S., Bianchi, N. O., Pantelias, G. E. &Wolff, S. (1985). The mechanism and pattern of banding induced by restriction endonucleases in human chromosomes.Chromosoma 91, 131–6.
Bianchi, N. O., Bianchi, M. S. &Cleaver, J. E. (1984). The action of ultraviolet light on the patterns of banding induced by restriction endonucleases in human chromosomes.Chromosoma 90, 133–8.
Bishop, D. T., Williamson, J. A. &Skolnick, M. H. (1983). A model for restriction fragment length distributions.Am. J. Hum. Genet. 35, 795–815.
Bullerdiek, J., Dittmer, J., Faehre, A., Bartnitzke, S., Kasche, V. &Schloot, W. (1985). A new baanding pattern of human chromosomes byin situ nick translation using EcoRI and biotin-dUTP.Clin. Genet. 28, 173–6.
Bullerdiek, J., Dittmer, J., Faehre, A. &Bartnitzke, S. (1986). Mechanisms ofin situ nick translation of chromosomes using restriction endonucleases.Cytobios 47, 33–44.
Burkholder, G. D. (1989). Morphological and biochemical effects of endonucleases on isolated mammalian chromosomesin vitro.Chromosoma 97, 347–55.
Gosden, J. R., Mitchell, A. R., Buckland, R. A., Clayton, R. P. &Evans, H. J. (1975). The location of four human satellite DNAs on human chromosomes.Exp. Cell Res. 92, 148–58.
Holmquist, G. (1988). DNA sequences in G-bands and R-bands. InChromosomes and Chromatin (edited byAdolph, K. W.) vol II, pp. 75–121. Boca Raton, Florida: CRC Press.
Kornberg, A. (1980).DNA Replication. San Francisco: W. H. Freeman & Co.
Lica, L. &Hamkalo, B. (1983). Preparation of centromeric heterochromatin by restriction endonuclease digestion of mouse L929 cells.Chromosoma 88, 42–9.
Lima-de-Faria, A., Isaksson, M. &Olsson, E. (1980). Action of restriction endonucleases on the DNA and chromosomes of Muntiacus muntjak.Hereditas 92, 267–73.
Lipchitz, L. &Axel, R. (1976). Restriction endonuclease cleavage of satellite DNA in intact bovine nuclei.Cell 9, 355–64.
Mezzanotte, R., Ferrucci, L., Vanni, R. &Bianchi, U. (1983a) Selective digestion of human metaphase chromosomes by Alu 1 restriction endonuclease.J. Histochem. Cytochem. 31, 553–6.
Mezzanotte, R., Bianchi, U., Vanni, R. &Ferrucci, L. (1983b). Chromatin organisation and restriction endonuclease activity on human metaphase chromosomes.Cytogenet. Cell. Genet. 36, 562–6.
Mezzanotte, R., Ferrucci, L., Vanni, R. &Sumner, A. T. (1985). Some factors affecting the action of restriction endonucleases on human metaphase chromosomes.Exp. Cell Res. 161, 247–53.
Mezzanotte, R., Vanni, R., Flore, O., Ferrucci, L. &Sumner, A. T. (1988). Ageing of fixed cytological preparations produces degradation of chromosomal DNA.Cytogenet. Cell Genet. 48, 60–2.
Mezzanotte, R., Peretti, D., Ennas, M. G., Vanni, R. &Sumner, A. T. (1989). Analysis of human metaphase chromosomes using antibodies to double-stranded and single-stranded DNA: staining patterns are related to DNA conformation.Cytogenet. Cell Genet. 50, 54–8.
Miller, D. A., Choi, Y-C. &Miller, O. J. (1983). Chromosome localization of highly repetitive human DNA's and amplified ribosomal DNA with restriction enzymes.Science 210, 395–7.
Millier, D. A., Gosden, J. R., Hastie, N. D. &Evans, H. J. (1984). Mechanism of endonuclease banding of chromosomes.Exp. Cell. Res. 155, 294–8.
Mitchell, A. R., Beauchamp, R. S. &Bostock, C. J. (1979). A study of sequence homologies in four satellite DNAs of man.J. Mol. Biol. 135, 127–49.
Prosser, J., Reisner, A. H., Bradley, M. L., Ho, K. &Vincent, P. C. (1981). Buoyant density and hybridisation analysis of human DNA sequences, including three satellite DNAs.Biochim. Biophys. Acta 656, 93–102.
Prosser, J., Frommer, M., Paul, C. &Vincent, P. C. (1986). Sequence relationships of three human satellite DNAs.J. Mol. Biol. 187, 145–55.
Viegas-Péquignot, E., Dutrillaux, B. &Thomas, G. (1988). Inactive X chromosome has the highest concentration of unmethylated Hha I sites.Proc. Natl. Acad. Sci. 85, 7657–60.
Waalwijk, C. &Flavell, R. A. (1978). Msp I, and isoschizomer of Hpa II which cleaves both unmethylated and methylated Hpa II sites.Nucl. Acids Res. 5, 3231–6.
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Sumner, A.T., Taggart, M.H., Mezzanotte, R. et al. Patterns of digestion of human chromosomes by restriction endonucleases demonstrated byin situ nick translation. Histochem J 22, 639–652 (1990). https://doi.org/10.1007/BF01047448
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DOI: https://doi.org/10.1007/BF01047448