Abstract
Metaphase chromosomes from the Chinese hamster cell line M3-1 were separated by means of a flow sorter. Two chromosome fractions were used for this study: A, which consisted of 95% pure chromosome no. 1, and B, which was 90% pure chromosome no. 2. The DNA of 106 chromosomes of each type was purified, and a 125I-cRNA transcript was synthesized in a reaction containing E. coli RNA polymerase and carrier-free 125I-CTP (1.7 Ci/μmole). The cRNA product synthesized with template DNA from 105 sorted chromosomes contained more than 106 dpm. The electrophoretic mobility profiles of the cRNAs on 7.5% SDS acrylamide gels demonstrated that more than 50% of the ribo-polymers were equal to or longer than marker E. coli met-tRNAf. In hybridization reactions 21% and 17% of the transcripts from Chinese hamster whole cell and sorted chromosome DNA hybridized to Chinese hamster DNA and did not hybridize significantly over background in reactions containing calf DNA at Crt values of 1.3 and 1.9×102 mole sec/l. Labelled cRNAs transcribed from the DNA of sorted chromosomes hybridized with the DNA of each sorted chromosome fractions at a Crt of 0.6 mole sec/l. This study demonstrated that the DNA can be (1) recovered from small numbers of highly purified flow sorted chromosomes, (2) used as template by E. coli RNA polymerase and (3) used to prepare a cRNA in reactions containing polymerase and carrier-free 125I-CTP to yield a product which can be employed for hybridization analysis.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Biessman, H., Gjerset, R.A., Levy, B.W., McCarthy, B.J.: Fidelity of chromatin transcription in vitro. Biochemistry 15, 4356–4363 (1976)
Burki, H.J., Regimbal, T.J., Jr., Mel, H.C.: Zonal fractionation of mammalian metaphase chromosomes and determination of their DNA content. Prep. Biochem. 3, 157–182 (1973)
Carrano, A.V., Gray, J.W., Moore, D.H., Minkler, J.L., Mayall, B.H., Dilla, M.A. van, Mendelsohn, M.L.: Purification of chromosomes of Indian muntjac by flow sorting. J. Histochem. Cytochem. 24, 348–354 (1976)
Commerford, S.L.: Iodination of nucleic acid in vitro. Biochemistry 10, 1993–2000 (1971)
Dube, S.K., Marcker, K.A., Clark, B.F.C., Cory, S.: Nucleotide sequence of N-formyl-methionyltransfer RNA. Nature (Lond.) 218, 232–233 (1968)
Gray, J.W., Carrano, A.V., Moore, D.H. II, Steinmetz, L.L., Minkler, J., Mayall, B.H., Mendelsohn, M.L., Dilla, M.A. van: High speed quantitative karyotyping by flow microfluorometry. Clin. Chem. 21, 1258–1262 (1975a)
Gray, J.W., Carrano, A.V., Steinmetz, L.L., Dilla, M.A. van, Moore, D.H. II, Mayall, B.H., Mendelsohn, M.L.: Chromosome measurement and sorting by flow systems. Proc. nat. Acad. Sci. (Wash.) 72, 1231–1234 (1975b)
Hanson, C.V.: Techniques in the isolation and fractionation of eukaryotic chromosomes. In: New techniques in biophysics and cell biology (R.H. Pain and B.J. Smith, eds.), p. 43–83. London: John Wiley and Sons 1975
Kissane, J.M., Robbins, E.: The fluorometric measurement of deoxyribonucleic acid in animal tissues with special reference to the nervous system. J. biol. Chem. 233, 184–188 (1958)
Kraemer, P., Deaven, L., Crissman, H., Dilla, M. van: Cell and molec. Biol. (E. Du Praw, ed.), pp. 47–109. New York: Academic Press 1972
Landel, A., Aloni, Y., Raftery, M.A., Attardi, G.: Electrofocusing analysis of HeLa cell metaphase chromosomes. Biochemistry 11, 1654–1662 (1972)
Loening, U.E.: The fractionation of high molecular weight ribonucleic acid by polyacrylamide gel electrophoresis. Biochem. J. 102, 251–257 (1967)
McCarthy, B.J.: The evolution of base sequences in polynucleotides. Progr. Nucleic Acid Res. and molec. Biol. 4, 129–160 (1965)
Miura, K.: Preparation of bacterial DNA by the pheno-pH 9-RNases method. In: Methods in enzymology 12, 543–545 (1967)
Salzman, N.P., Moore, D.E., Mendelsohn, J.: Isolation and characterization of human metaphase chromosomes. Proc. nat. Acad. Sci. (Wash.) 56, 1449–1456 (1966)
Sawin, V.L., Scherberg, N., Carrano, A.: Hybridization in situ of 125I-cRNA transcribed from sorted metaphase chromosomes. Proc. 1977 ICN-UCLA Symp. on molec. and cell. Biol. Academic Press (in press, 1978)
Scherberg, N.H., Refetoff, S.: The preparation of carrier-free iodine isotope-substituted cytosine nucleotides. Biochim. biophys. Acta (Amst.) 340, 446–451 (1974)
Stubblefield, E., Deaven, L., Cram, L.S.: Flow microfluorometric analysis of isolated Chinese hamster chromosomes. Exp. Cell Res. 94, 464–468 (1975)
Stubblefield, E., Wray, W.: Biochemical and morphological studies of partially purified Chinese hamster chromosomes. Cold Spr. Harb. Symp. quant. Biol. 38, 835–843 (1973)
Walker, P.M.B.: The specificity of molecular hybridization in relation to studies on higher organisms. Progr. Nucleic Acid Res. and molec. Biol. 9, 301–326 (1969)
Wray, W.: Isopycnic centrifugation of mammalian metaphase chromosomes in metrizamide. FEBS Lett. 62, 202–207 (1967)
Wray, W., Stubblefield, E.: A new method for the rapid isolation of chromosomes, mitotic apparatus of nuclei from mammalian fibroblasts at near neutral pH. Exp. Cell Res. 59, 469–478 (1970)
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Sawin, V.L., Rowley, J.D. & Carrano, A.V. Transcription and hybridization of 125I-cRNA from flow sorted chromosomes. Chromosoma 70, 293–304 (1979). https://doi.org/10.1007/BF00328767
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00328767