The Use of Cell Fusion in the Analysis of Gene ActionAuthor(s): Henry HarrisSource: Proceedings of the Royal Society of London. Series B, Biological Sciences, Vol. 176, No.1044, A Discussion on Gene Action and its Control (Dec. 1, 1970), pp. 315-317Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/75926 .

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Proc. Roy. Soc. Lond. B. 176, 315--317 (1970)

Printed in Great Britain

The use of cell fusion in the analysis of gene action (Summary only)

BY HENRY HARRIS, F.R.S.

Sir William Dunn School of Pathology, South Parks Road, Oxford, OX 13 RE

When, under the influence of inactivated Sendai virus, the nucleus of a mature hen

erythrocyte is introduced into the cytoplasm of a human or mouse tissue culture

cell, it resumes the synthesis of RNA and DNA. This reactivation of the red cell nucleus in the heterokaryon is associated with a marked increase in its volume. There is a direct relation between the volume of the nucleus and the amount of RNA that it makes. The nuclear enlargement is not the consequence of increased RNA synthesis, or of DNA synthesis: enlargement is the primary event, and the increase in RNA synthesis is determined by it. During the process of reactivation, the erythrocyte nucleus shows a five- to six-fold increase in dry mass which takes

place largely before the replication of DNA begins. This increase is due to the

passage of cytoplasmic proteins into the erythrocyte nucleus. The physical properties of the deoxyribonucleoprotein complex in the erythrocyte nucleus

change as the nucleus enlarges. The ability of the nuclear chromatin to bind acri- dine orange and other intercalating dyes increases four- to fivefold; and changes in the melting profile of the deoxyribonucleoprotein indicate that its structure is loosened. It appears that, as the nucleus expands, more of the chromatin passes from a condensed to a dispersed state and more of it is transcribed.

At the concentrations used to induce cell fusion, Sendai virus is haemolytic and

rapidly lyses the nucleated erythrocytes. Fusion then takes place between the other cells in the combination and erythrocyte ghosts. The erythrocyte nucleus is thus introduced into the cytoplasm of the recipient cell without any appreciable contribution of erythrocyte cytoplasm. The reactivation of the hen erythrocyte nucleus is therefore achieved by signals emanating from human or mouse cyto- plasm.

For at least 2 or 3 days after cell fusion, the reactivated erythrocyte nuclei do not develop nucleoli that can be discerned under the light microscope. During this

period, the heterokaryon, although it may contain several active erythrocyte nuclei, does not synthesize any hen-specific surface antigens; and the hen-specific antigens introduced into the surface of the heterokaryon by the process of cell fusion are eliminated. But when, later, the erythrocyte nuclei do develop nucleoli, hen-specific antigens reappear on the surface of the heterokaryon and progres- sively accumulate. When embryonic chick erythrocyte nuclei are reactivated in the heterokaryon, nucleoli appear more rapidly, and chick-specific antigens reappear on the surface of the cell sooner. There is a close correlation under all conditions

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Henry Harris (Discussion Meeting)

between the time at which the nucleoli appear in the erythrocyte nuclei and the time at which hen-specific antigens reappear on the surface of the cell.

When a chick erythrocyte nucleus is introduced into the cytoplasm of a mutant mouse cell lacking the enzyme inosinic acid pyrophosphorylase, synthesis of chick inosinic acid pyrophosphorylase is induced. However, this synthesis does not begin until the erythrocyte nucleus develops a nucleolus. The kinetics of

synthesis of this soluble enzyme are essentially similar to those found for the

synthesis of the surface antigens specified by the chick nucleus. Before developing nucleoli, the erythrocyte nuclei synthesize little, if any,

normal 28s or 16s RNA, but they do synthesize large amounts of the RNA that shows polydisperse sedimentation in conventional sucrose gradients. Autoradio-

graphic studies involving the use of a microbeam of ultraviolet light show, how- ever, that this 'polydisperse' RNA is not transferred to the cytoplasm of the cell in detectable amounts so long as the erythrocyte nucleus lacks a definitive nucleolus. The inability of the erythrocyte nucleus at this stage to determine the synthesis of

chick-specific surface antigen or chick-specific soluble enzyme is thus attributable to the fact that it fails to transfer the RNA made on its chromosomes to the cyto- plasm of the cell. The pre-existing ribosomes in the cytoplasm of the heterokaryon cannot achieve the transfer of genetic information from the 'anucleolate' erythro- cyte nucleus. When the erythrocyte nuclei develop nucleoli, however, the RNA which they make is transferred to the cytoplasm of the cell and the synthesis of

chick-specific antigen and the enzyme then begins. A similar effect is seen in normal mononucleate cells when the nucleolus alone is

inactivated by a microbeam of ultraviolet light. Inactivation of the nucleolus in this

way inhibits the transfer to the cytoplasm, not only of the RNA synthesized at the nucleolar site, but also of the RNA made elsewhere in the nucleus.

These experiments permit the following conclusions: (1) Regulation of the

transcription of DNA in animal cell nuclei is associated with condensation and

dispersion of the chromatin. Inactivation and reactivation of nuclei is linked to

changes in nuclear volume. (2) The signals which govern transcription of the DNA

operate across wide species differences. (3) There is no mandatory, or even close, coupling between transcription and translation. In reactivated erythrocyte nuclei, many genes may be transcribed for several days without determining the synthesis of any proteins. (4) The process of switching genes on in a dormant nucleus is a

relatively slow one extending over days rather than hours. Genetically identical nuclei in the same cytoplasm may be switched on at very different rates, and these rates are determined not by differences in the nature of the cytoplasmic signals, but by differences in the physiological state of the nuclei. (5) The RNA made in the nucleolus and the RNA made elsewhere in the nucleus are not transferred to the

cytoplasm independently of each other. A nucleus does not transfer genetic informa- tion to the cytoplasm of the cell until it develops its own nucleolus and begins to

synthesize its own ribosomal RNA.

316

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Cell fusion in the analysis of gene action 317

REFERENCES (Harris) Bolund, L., Ringertz, N. R. & Harris, H. I969 J. Cell Sci. 4, 71. Cook, P. R. 1970 J. Cell Sci. 7, 1. Harris, H. I967 J. Cell Sci. 2, 23. Harris, H. & Cook, P. R. I969 J. Cell Sci. 5, 121. Harris, H., Sidebottom, E., Grace, D. M. & Bramwell, M. E. I969 J. Cell Sci. 4, 499. Harris, H. & Watkins, J. F. I965 Nature, Lond. 205, 640. Harris, H., Watkins, J. F., Ford, C. E. & Schoefl, G. . 1966 J. Cell Sci. 1, 1. Schneeberger, E. E. & Harris, H. I966 J. Cell Sci. 1, 401. Sidebottom, E. & Harris, H. I969 J. Cell Sci. 5, 351.

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