The next candidate to be considered was DNA polymerase, an enzyme that promotes the incorporation of precursor substances into new DNA in a way that is specified by the composition of the preexisting "template" DNA. DNA polymerase activity in living cells has been tested by introducing purified DNA and tritium-labeled thymidine into eggs. In collaboration with Max Birnstiel of the University of Edinburgh we have established that the injected DNA serves as a template for synthesis of the same kind of DNA. When DNA and labeled thymidine are introduced into oocytes, no DNA replication can be detected. This means that the cytoplasmic factor in ducing DNA synthesis in eggs includes DNA polymerase or something that ac tivates this enzyme. It is doubtful, how ever, that this is the only constituent of the inducer. If it were, the injection of egg cytoplasm (which contains DNA polymerase) into oocytes might be expected to induce DNA synthesis, a result that is not in fact obtained. This experiment, in which purified DNA is replicated in the cytoplasm of unfertilized eggs, also serves to demonstrate that constituents of injected brain nuclei other than their DNA are not required in order to initiate the particular reaction being discussed here.
The last aspect of this reaction on which some information is available concerns the mechanism by which the inducing factors in the cytoplasm interact with the DNA in the nucleus. It was noticed several years ago by Stephen Subtelny, now at Rice University (and subsequently by others), that transplanted nuclei increase in volume soon after they have been injected into eggs. A pronounced swelling is also observed in nuclei injected into oocytes; the swelling is therefore not directly related to a particular type of nuclear response. During this nuclear enlargement chromatin (which contains the genetic material in the nucleus) becomes dispersed and, as Arms has demonstrated, cytoplasmic protein also enters the swelling nuclei. While working at Oxford, Robert W. Merriam of the State University of New York at Stony Brook found a close temporal relation between the passage of cytoplasmic protein into enlarging nuclei and the initiation of DNA synthesis. The interpretation of these events currently favored by those of us involved in the experiments is that the nuclear swelling and chromatin dispersion facilitate the association of cytoplasmic regulatory molecules with chromosomal genes, thereby leading to a change in gene activity of a kind determined by the nature of the molecules that enter the nucleus.
The experiments described here have established two general conclusions. First, nuclear genes are not necessarily lost or permanently inactivated in the course of cell differentiation. Second, major changes in chromosome function as well as in different kinds of gene activity can be experimentally induced by normal constituents of living cell cytoplasm. The same type of experiment is now proving useful in attempts to determine the identity of the cytoplasmic components and their mode of action.
We have had to restrict our attention to what can be described as sequential changes in gene activity, that is, differences between one developmental stage and the next. These may be compared with regional variations in nuclear activity, that is, differences between one part of an embryo and another at the same developmental stage. The latter are hard to study biochemically because of the difficulty in obtaining enough material. There is no obvious reason, however, why the processes leading to the two types of differentiation should be fundamentally different.
Experiments analogous to those described here have been conducted with bacteria infected with viruses, with nuclear transplantation in protozoans and with fusion in mammalian cells. Each kind of material is well suited for certain problems; nuclear transplantation utilizing amphibian eggs and cell nuclei is especially suited to the analysis of processes that lead to the first major differences between cells. Only after these differences have been established by constituents of egg cytoplasm are cells able to respond differentially to the important agents that guide development, such as inducer substances and hormones. Finally, the technique of nuclear transplantation may be used to introduce cell components other than the nucleus into the cytoplasm of different living cells; this is likely to be of great value for the more detailed analysis of early development and cell differentiation.