Cytogenetics / edited by Ronald L. Phillips, Charles R. Burnham.

Date:
[1977], ©1977
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Licence: Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)

Credit: Cytogenetics / edited by Ronald L. Phillips, Charles R. Burnham. Source: Wellcome Collection.

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    49 Reprinted from Natl. Acad. Sci. (U.S.A.) Proc. 63(2):378-383 (1%9) FORMATION AND DETECTION OF RNA-DNA HYBRID MOLECULES IN CYTOLOGIC AL PREPARATIONS* By Joseph G. Gall and Mary Lou Pardue KLINE BIOLOGY TOWER, YALE UNIVERSITY Communicated by Normxin H. Giles, March 27, 1969 Abstract.—A technique is described for forming molecular hybrids between RNA in solution and the DNA of intact cytological preparations. Cells in a conventional tissue squash are immobilized under a thin layer of agar. Next they are treated with alkali to denature the DNA and then incubated with tritium-labeled RNA. The hybrids are detected by autoradiography. The technique is illustrated by the hybridization of ribosomal RNA to the amplified ribosomal genes in oocytes of the toad Xenopus. A 1олу level of gene amplifica¬ tion was also detecjbed in premeiotic nuclei (oogonia). Several techniques are currently used for annealing RNA molecules to their complementary DNA sequences. For certain purposes both the RNA and DNA can be in solution,^- ^ but it is often more convenient to have the DNA im¬ mobilized in a solid or semisolid matrix,®- * or attached to a nitrocellulose mem¬ brane filter.® The hybrids are generally detected by scintillation counting of radioactive RNA after treatment with ribonuclease to remove unhybridized RNA. The hybridization of RNA to the DNA in a cytological preparation should exhibit a high degree of spatial localization, since each RNA species hybridizes only with sequences to which it is complementary. The general principles of a cytological hybridization technique are not difficult to lay down. The chromo¬ somes or nucleus should be fixed in as lifelike a fashion as possible; basic proteins should be removed, since they are known to interfere with the hybridization procedure;® the DNA should be denatured in such a way that cytological integ¬ rity is not lost; the hybridization should be carried out with radioactive RNA of very high specific activity, since the number of hybridized molecules at a given locus will be small; and detection should be by tritium autoradiography to permit maximal cytological resolution. This communication describes a cytological hybridization technique apphcable to conventional squash preparations. It is illustrated by the hybridization of rRNA to the extrachromosomal rDNA in oocytes of the toad Xenopus. A pre¬ liminary report on the technique was presented in December 1968 at the Inter¬ national Symposium of Nuclear Physiology and Differentiation, Belo Horizonte, Brazil.® Materials and Methods.—The cytological hybridization technique combines certain features of the agar column* and filter methods.® It should be generally applicable to any material that can be examined as a squash or smear. The following procedure was used in making the preparation shown in Figure 1. (1) Ovaries from recently metamorphosed Xenopus laevis were fixed for a few minutes in ethanol-acetic acid (3:1). (2) The tissue was transferred to a drop of 45% acetic acid on a microscope slide and 471