The next set of experiments was designed to find out if cytoplasmic components can repress or activate genes, that is, if they can select which genes in a nucleus will be active at any one time. Advantage was taken of the natural dissociation that exists in the time of synthesis of different classes of RNA during the early embryonic development of Xenopus. The work of several investigators has established the following sequence of events in Xenopus embryos. For the first 10 divisions after fertilization no nuclear RNA synthesis can be detected. Just after this—at the mid-late blastula stage—the cells synthesize large RNA molecules, which are believed not to include ribosomal RNA but which are likely to include "messenger" RNA. Toward the end of the blastula stage "transfer" RNA synthesis is first detected; this is followed a few hours later, during the formation of the gastrula, by the synthesis of ribosomal RNA.
The extent to which these events are under cytoplasmic control has been investigated by transplanting into enucleated eggs single nuclei from embryonic tissue at the neurula stage of development, the one that follows the gastrula stage. As the nuclear-transplant embryos develop, RNA precursor substances that have been labeled with radioactive atoms (for example uridine triphosphate labeled with tritium, the radioactive form of hydrogen) are used to determine the classes of RNA being synthesized at each stage. Autoradiography has shown that a neurula nucleus, which synthesizes each main kind of RNA, stops all detectable RNA syntheis, that is, it no longer incorporates labeled RNA precursors, within an hour of transplantation into egg cytoplasm. Furthermore, chromatography and other kinds of analysis show that, when the transplant embryos are reared through the blastula and gastrula stages, they synthesize heterogeneous RNA, transfer RNA and ribosomal RNA in turn and in the same sequence as do embryos reared from fertilized eggs.
Taken together, these experiments have shown that changes in the type of gene product (for example the synthesis of RNA or DNA), as well as changes in the selection of genes that are active (for example the synthesis of different types of RNA), can be experimentally induced. Since a high proportion of transplanted neurula nuclei support entirely normal development, the results show that egg cytoplasm must contain constituents responsible for independently controlling the activity of different classes of genes in normal living nuclei.
We can now consider what is perhaps the most interesting question of all: What is the mechanism by which cytoplasmic components bring about changes in gene activity? Of the various changes in chromosome and gene activity that can be experimentally induced in transplanted nuclei, special attention has been devoted to the induction of DNA synthesis by egg cytoplasm. It is easier to analyze than other changes, and it seems likely to exemplify certain general principles of cytoplasmic regulation in early embryonic development.
The origin of the cytoplasmic condition that induces DNA synthesis has been investigated by injecting adult brain nuclei, together with a radioactive labeling substance, into growing and maturing oocytes. The inducing factor appears just after an increase in the level of pituitary hormone has caused an oocyte to mature into an egg, an event that is accompanied by intensive RNA and protein synthesis.
Concerning the identity of the inducing factor, the first candidate to be considered was simply the presence of an adequate supply of DNA precursor substances. Woodland, however, has injected growing oocytes with 10 times the amount of all four common DNA precursors believed to be present in the mature egg. One of the precursors, thymidine triphosphate, had been labeled with tritium. In spite of the availability of these precursors, the brain nuclei did not incorporate the labeled thymidine into DNA. Although this experiment requires further analysis before DNA precursors can be excluded as inducers of DNA synthesis, it encourages a search in other directions.