Abstract
A microfluorimetric method has been developed for determination of DNA content in individual human chromosomes. The method is based on a preliminary identification of chromosomes with Hoechst 33258 followed by staining of the chromosomes with Feulgen reaction by using Schiff’s reagent type ethidium bromide-SO2 and then by measuring the fluorescence intensity of the chromosomes by using an image analyzer. The method allows determining the DNA content of individual chromosomes with an accuracy up to 4.5 fg. The DNA content of individual human chromosomes and their p-and q-arms, as well as homologous chromosomes, were measured by using the developed method. It has been shown that the DNA content in chromosomes of the normal human karyotype is unstable and can fluctuate in some chromosomes within 35–40 fg.
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Agafonova, N.A., Ushal, I.E., and Kudryavtsev, B.N., Method of DNA television fluorometry in individual human chromosomes, Tsitologiia, 2002, vol. 44, no. 9, p. 858.
Agroskin, L.S. and Papayan, G.V., Tsitofotometriya (Cytophotometry), Leningrad: Nauka, 1977.
Antonarakis, S.E., Human genome sequence and variation, in Human Genetics: Problems and Approaches, Berlin: Springer-Verlag, 2010, vol. 2, pp. 31–53.
Arnheim, N., Krystal, M., Schmickel, R., Wilson, G., Ryder, O., and Zimmer, E., Molecular evidence for genetic exchanges among ribosomal genes on nonhomologous chromosomes in man and apes (restriction maps/concerted evolution/unequal crossing-over/natural selection/chromosome abnormalities), Proc. Natl. Acad. Sci. USA, 1980, vol. 77, pp. 7323–7327.
Baranov, V.S., Tsitogenetika embrional’nogo razvitiya cheloveka: nauchno-prakticheskie aspekty (Cytogenetics of Human Embryonic Development: Scientific and Practical Aspects), St. Petersburg: N.-L., 2007.
Beneke, G., Application of interference microscopy to biological material, in Vvedenie v kolichestvennuyu tsitokhimiyu (Introduction to Quantitative Cytochemistry), Wied, G.L. and Bahr, G.F., Eds., New York: Academic Press, 1966.
Bosman, F., Van, Der, Ploeg, M., and Geraedts, J.P.M., Influence of Q and G-banding on the Feulgen-stainability of human metaphase chromosomes, Histochem. J., 1977, vol. 9, pp. 31–33.
Brodskii, V.Ya., Results and prospects of quantitative cytochemical studies, in Vvedenie v kolichestvennuyu tsitokhimiyu (Introduction to Quantitative Cytochemistry), Wied, G.L. and Bahr, G.F., Eds., New York: Academic Press, 1966.
Chakraborty, B.M., and Chakraborty, R., Variations in the length of human Y chromosome: a statistical study, Acta Anthropogenet., 1984, vol. 8, nos. 3–4, pp. 269–275.
de, Carvalho, C.M.B. and Santos, F.R., Human Y-chromosome variation and male dysfunction, J. Mol. Genet. Med., 2005, vol. 1, pp. 63–75.
Doležel, J., Bartoš, J., Voglmayr, H., and Greihuber, J., Nuclear DNA content and genome size of trout and human, Cytometry A, 2003, vol. 51A, pp. 127–128.
Donehower, L., Furlongs, C., Gillespie, D., and Kurnit, D., DNA Sequence of baboon highly repeated DNA: evidence for evolution by nonrandom unequal cross-overs (satellite dna/nucleosome/heterochromatin/primates, Proc. Natl Acad. Sci. USA, 1980, vol. 77, pp. 2129–2133.
Gaillard, J.L.I., Van, Duiyh, and Schaberg, A., Photometric determination of DNA in human chromosomes, Exp. Cell Res., 1968, vol. 53, pp. 417–431.
Graham, K.A., Richardson, C.L., Minden, M.D., Trent, J.M., and Buick, R.N., Varying degrees of amplification of the N. ras oncogene in the human breast cancer cell line MCF-7, Cancer Res., 1985, vol. 45, pp. 2201–2205.
Gregory, T.R., Coincidence, coevolution, or causation? DNA content, cell size, and the C-value enigma, Biol. Rev. Camb. Philos. Soc., 2001, vol. 76, pp. 65–101.
Grinchuk, T.M., Sukhikh, T.R., Sorokina, E.A., Artsybasheva, I.V., Pan’shina, Yu.T., Rozanov, Yu.M., Kudryavtsev, B.N., and Ignatova, T.N., Microfluorimetric estimate of the DNA content of individual chromosomes confirms the cytogenetically detected DNA amplification in structural variants of chromosome 1 in Chinese hamster cells with a stable multiple resistance, Doklady Akad. Nauk SSSR, 1986, vol. 286, no. 3, pp. 712–717.
Grinchuk, T.M., Ushal, I.E., Artsybasheva, I.V., Pavlenko, M.A., and Kudryavtsev, B.N., Microfluorimetric analysis of dynamics of genomic changes of Chinese hamster fibroblasts CHL V-79 RJK with multidrug resistance, Tsitologiia, 2007, vol. 49, no. 12, pp. 1011–1015.
Gripenberg, U., Size variation and orientation of the human Y chromosome, Chromosoma, 1964, vol. 15, pp. 618–629.
Groen, F.C.A., and van, der, Ploeg, M., DNA cytophotometry of human chromosomes, J. Histochem. Cytochem., 1979, vol. 27, pp. 436–440.
Hardie, D.C., Gregory, T.R., and Hebert, P.D., From pixels to picograms: a beginners’ guide to genome quantification by Feulgen image analysis densitometry, J. Histochem. Cytochem., 2002, vol. 50, pp. 735–749.
Harris, P., Boyd, E., Young, B.D., and Ferguson-Smith, M.A., Determination of the DNA content of human chromosomes by flow cytometry, Cytogenet. Cell Genet., 1986, vol. 41, pp. 14–21.
Karpishchenko, A.I., Meditsinskie laboratornye tekhnologii (Medical laboratory technologies), St. Petersburg: Nauka, 1999.
Khesin, R.B., Nepostoyanstvo genoma (Variability of the Genome), Moscow: Nauka, 1985.
Krishan, A., Dandekar, P., Nathan, N., Hamelik, R., Miller, C., and Shaw, J., DNA index, genome size, and electronic nuclear volume of vertebrates from the Miami Metro zoo, Cytometry A, 2005, vol. 65A, pp. 26–34.
Kudryavtsev, B.N., Kudryavtseva, M.V., and Faddeeva, M.D., DNA cytofluorimetry with ethidium bromide-SO-2, Tsitologiia, 1974b, vol. 16, no. 1, pp. 107–111.
Kudryavtsev, B.N., Kudryavtseva, M.V., and Rozanov, Yu.M., Determination of optimal conditions for cytochemical tests for microfluorometric determination of DNA in cells. IV. The differentiation step in the Feulgen test with auramine-SO-2, Tsitologiia, 1974a, vol. 16, no. 2, pp. 256–261.
Lazyuk, G.I., Teratologiya cheloveka (Human Teratology), Moscow: Meditsina, 1991.
Leslie, I., The nucleic and content of tissues and cells, in The Nucleic Acids, New York: AP Inc., 1955, vol. 2, pp. 1–44.
Litvinchuk, S.N., Rozanov, Yu.M., Borkin, L.Ya., and Skorinov, D.V., Molecular-biochemical and cytogenetic aspects of microevolution in tailless amphibians of the fauna of Russia and adjacent countries, in Materialy III s”ezda Gerpetologicheskogo obschestva im. A. M. Nikol’skogo (Proceedings of the III Congress of the A.M. Nikol’skii Herpetological Society), Pushchino., 2008, pp. 247–257.
Litvinchuk, S.N., Rozanov, Yu.M., Usmanova, N.M., Borkin, L.Ya., Mazanaeva, L.F., and Kazakov, V.I., Variation of microsatellites BM224 and Bcal7 in populations of green toads (Bufo viridis complex) with various nuclear DNA content and ploidy, Tsitologiia, 2006, vol. 48, no. 4, pp. 332–345.
Lyapunova, N.A., Egolina, N.A., and Mkhitarova, E.V., Interindividual and intercellular differences in the total activity of ribosomal genes detectable by the Ag staining of nucleolus organizer regions in human acrocentric chromosomes, Russ. J. Genet., 1988, vol. 24, no. 7, pp. 1282–1288.
Lyckegaard, E.M., and Clark, A.G., Ribosomal DNA and stellate gene copy number variation on the Y chromosome of Drosophila melanogaster, Proc. Natl. Acad. Sci. USA, 1989, vol. 86, pp. 1944–1948.
Macgregor, H.C. and Varley, J, Working with animal chromosomes, Chichester, Wiley, 1984.
Mamaeva, S.E., Atlas khromosom postoyannykh kletochnykh liny cheloveka i zhivotnykh (Atlas of chromosomes of permanent cell lines of humans and animals), Moscow: Nauchnyi mir, 2002.
Mattson, P., and Rydberg, B., Analysis of chromosomes from human peripheral lymphocytes by flow cytometry, Cytometry, 1981, vol. 1, pp. 369–372.
McKay, R.D.G., Bobrow, M., and Cooke, H.J., The identification of a repeated DNA sequence involved in the karyotype polymorphism of the human Y chromosome, Cytogenet. Cell Genet., 1978, vol. 21, pp. 19–32.
McKenzie, W.H., Hostetter, T.L., and Lubs, H.A., Y Family study: heritable variation in the length of the human Y chromosome, Am. J. Hum. Genet., 1972, vol. 24, pp. 686–693.
Mefford, H., van, den, Engh, G., Friedman, C., and Trask, B.J., Analysis of the variation in chromosome size among diverse human populations by bivariate flow karyotyping, Hum. Genet., 1997, vol. 100, pp. 138–144.
Mendelsohn, M.L., Mayall, B.H., Bogart, E., Moore, D.H., and Perry, B.H., DNA content and DNA-based centromeric index of the 24 human chromosomes, Science, 1973, vol. 179, pp. 1126–1129.
Mirsky, E., and Ris, H., The desoxyribonucleic acid content of animal cells and its evolutionary significance, J. Gen. Phiysiol., 1951, vol. 34, pp. 451–462.
Ornstein, L., Maunter, W., Davis, B.J., and Tamura, R., New horizons in fluorescence microscopy, J. Maunt. Sinai Hospt., 1957, vol. 24, pp. 1066–1078.
Prokof’eva-Bel’govskaya, A.A., Geterokhromaticheskie rayony khromosom (Heterochromatic regions of chromosomes), Moscow: Nauka, 1986.
Rasch, E.M., Barr, H.J., and Rasch, R.W., The DNA content of sperm of Drosophila melanogaster, Chromosoma, 1971, vol. 33, pp. 1–18.
Rasch, E.M., DNA “standards” and the range of accurate DNA estimates by Feulgen absorption, Prog. Clin. Biol. Res., 1985, vol. 196, pp. 137–66.
Rozanov, Yu.M. and Vinogradov, A.E., Precise DNA cytometry: investigation of individual variability in animal genome size, Tsitologiia, 1998, vol. 40, no. 8/9, pp. 792–799.
Rozanov, Yu.M., Kudryavtsev, B.N., and Kudryavtseva, M.V., On the accuracy and sensitivity of the flow cytometry method, in Vsesoyuznyi simpozium “Tsitokhimicheskie aspekty razvitiya i funktsionirovaniya nervnoy sistemy” (All-Union Symposium “Cytochemical Aspects of the Development and Functioning of the Nervous System”), Tbilisi, 1976, pp. 69–71.
Rozanov, Yu.M., Litvinchuk, S.N., Borkin, L.Ya., and Chagin, V.O., The study of animal genome size variation: evidence of the existence of 1 Mb chromatin epidomain, Tsitologiia, 2007, vol. 49, no. 9, pp. 788.
Rudkin, G.T., Photometric measurements of individual metaphase chromosomes, In Vitro, 1965, vol. 1, pp. 12–20.
Shapiro, H.S., Deoxyribonucleic acid content per cell of various organisms, in Handbook of Biochemistry and Molecular Biology, Cleveland: CRC Press, 1976, vol. 2, pp. 284–306.
Shtein, G.I., Panteleev, V.G., Povarkova, A.V., and Kudryavtsev, B.N., Potential of the image analyzer “Videotest” for performing microphotometric study in cytology, Tsitologiia, 1998, vol. 40, no. 10, pp. 913–916.
Stults, D.M., Killen, M.W., Pierce, H.H., and Piercé, A.J., Genomic architecture and inheritance of human ribosomal RNA gene clusters, Genome Res., 2008, vol. 18, pp. 13–18.
Sukhikh, T.R., DNA microfluorometry in individual mammalian chromosomes, Extended Abstract of Cand. Sci. (Biol.) Dissertation, Leningrad, 1985.
Sukhikh, T.R., Kalimagambetov, A.M., Barskii, I.Ya., Khomenkova, S.A., Vinogradov, A.E., Rozanov, Yu.M., and Kudryavtsev, B.N., A photographic microfluorimetric method for determining the DNA content in individual human chromosomes, Tsitologiia, 1989, vol. 31, no. 11, pp. 1329–1337.
Tiersch, T.R., Chandler, R.W., Wachtel, S.S., and Elias, S., Reference Standards for flow Cytometry and Application in Comparative Studies of Nuclear DNA Content, Cytometry, 1989, vol. 10, pp. 706–710.
Tusie-Luna, M-T. and White, P.C., Gene conversions and unequal crossovers between CYP21 (steroid 21-hydroxylase gene) and CYP21P involve different mechanisms (recombination/polymerase chain reaction/spermatozoa), Proc. Natl. Acad. Sci. USA, 1995, vol. 92, pp. 10796–10800.
Unne’rus, V., Fellman, J., and de, la, Chapelle, A., The length of the human Y chromosome, Cytogenetics, 1967, vol. 6, pp. 213–227.
Veiko, N.N., Shubaeva, N.O., Malashenko, A.M., Beskova, T.B., Agapova, R.K., and Lyapunova, N.A., Ribosomal genes in inbred mouse strains: interstrain and intrastrain variation of copy number and extent of methylation, Russ. J. Genet., 2007, vol. 43, no. 9, pp. 1021–1031.
Vendrely, R. and Vendrely, C., The results of cytophotometry in the study of the deoxyribonucleic acid (DNA) content of the nucleus, Int. Rev. Cytol., 1956, vol. 5, pp. 171–194.
Vinogradov, A.E., Genome size and GC-percent in vertebrates as determined by flow cytometry: the triangular relationship, Cytometry, 1998, vol. 31, pp. 100–109.
Vogel, F. and Motulsky, A.G., Human Genetics, Berlin: Springer-Verlag, 1987.
Watson, D., Molecular Biology of the Gene, 3rd ed., San Francisco: Benjamin-Cummings Publ., 1976.
Zakharov, A.F., Benyush, V.A., Kuleshov, N.P., and Baranovskaya, L.I., Khromosomy cheloveka (Human Chromosomes), Meditsina, 1982.
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Original Russian Text © N.A. Agafonova, G.A. Sakuta, Yu.M. Rozanov, G.I. Shtein, B.N. Kudryavtsev, 2013, published in Tsitologiya, 2013, Vol. 55, No. 5, pp. 338–347.
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Agafonova, N.A., Sakuta, G.A., Rozanov, Y.M. et al. DNA image-fluorimetry of individual human chromosomes. Cell Tiss. Biol. 7, 352–361 (2013). https://doi.org/10.1134/S1990519X13040020
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DOI: https://doi.org/10.1134/S1990519X13040020