When Spiegelman transferred to Washington University in 1942, he chose as his doctoral thesis topic an obscure phenomenon then known as enzymatic adaptation, subsequently renamed enzyme induction. It was known that the ability of a given microorganism (e.g., yeast) to manufacture various enzymes--the special proteins that catalyze the thousands of chemical reactions in living systems--could be used to track genetic changes in these microbes. Changes in a microbe's repertoire of enzymes would indicate a change in its genes, i.e., a mutation. However, since the 1880s, researchers had observed that a population of microbial cells, placed in contact with some substrate (e.g., a nutrient such as lactose or galactose) could sometimes produce the enzyme needed to break down and digest that substrate, even if it was a substance not normally consumed by the microbe. This change in the enzyme system appeared to be heritable, but the microbial offspring did not seem to be mutants. Spiegelman was skeptical, and suspected that what was actually happening was mutation followed by selection, in keeping with the principle of evolution. George Beadle and Edward Tatum had just reported their discovery that genes controlled the synthesis of enzymes--one gene to one enzyme--so that synthesis of an enzyme would indicate a corresponding gene in the organism. However, Spiegelman realized that if the adaptation phenomenon could be validated, it would furnish a way to study enzyme formation within a stable genetic situation, rather than by tracking mutations. It would then be possible to explore a number of important questions about how enzymes are formed in cells, how far and in what directions the enzymatic make-up of a cell with a given genome could be modified experimentally, and how this enzymatic variation depended on the genome.
Spiegelman pursued these questions for over a decade. Initially he worked with strains of Saccharomyces cerevisiae, a type of yeast for which enough genetic markers existed to identify mutants. The enzymes studied were galactosidase and melibiase, which metabolize, respectively, the sugars galactose and melibiose. He found that some strains could adapt only via mutation, but that others could indeed do so without changing their genes, induced by exposing them to simple nutrient substances. Thus, the expression of yeast genes for making various enzymes could be "turned off and on" with the aid of inducers. With these methods researchers could begin to analyze the mechanisms controlling gene-enzyme relationships. Spiegelman was awarded his PhD in 1944 on the basis of this work.
As an assistant professor at Washington University during this time, he found that the yeast enzymes, once formed, were capable of self-duplication without intervention from the gene; the gene's function seemed to be only to initiate the synthesis of the first few enzyme molecules. The duplication was occurring outside the cell nucleus, in the cytoplasm. Some years earlier, geneticist Sewell Wright had proposed that adaptation might occur via partial or complete copies of genes located in the cytoplasm that served as the programs for protein synthesis, and Spiegelman's research seemed to give weight to this idea. Using a radioactive phosphorus tracer in the growing yeast's proteins, he found that proteins normally associated with the cell nucleus migrated to the cytoplasm. Spiegelman's collaborator Carl Lindegren called these "cytogenes"--eventually they were termed "plasmagenes." This suggestion anticipated by over a decade the "messenger" concept of François Jacob and Jacques Monod, which later led to the discovery of messenger RNA as the transmitter of genetic information from DNA.
At the time, Spiegelman believed that his findings could have application to the problem of cancer. If certain genes for enzymes could be activated or deactivated, perhaps something similar was occurring with the genes governing normal reproductive functions in tissue cells, causing the characteristic rapid proliferation. He and several of his colleagues were profiled in the press as young scientists poised to discover a cure for cancer. The possible application of the enzyme work to cancer research brought him generous funding from the American Cancer Society, administered through a U.S. Public Health Service research fellowship. Spiegelman chose to spend the fellowship year at the University of Minnesota. The funding was not sustained, however, and in 1949 he accepted a post as professor of microbiology at the University of Illinois, where he continued his enzyme induction studies.
The earlier work that Spiegelman and others had done made it clear that the presence of a particular gene in a cell's nucleus did not guarantee that the corresponding enzyme would be found in the cytoplasm in measurable amounts. Thus, it was impossible to predict phenotype (physical characteristics) from genotype alone. Beadle and Tatum's statement that genes control enzyme synthesis had to be revised to say that genes control the potentiality of enzyme synthesis. Spiegelman went on to investigate how this potential was made actual. Did cells contain enzyme precursor substances that could be readily converted into active enzyme? No evidence of precursors could be found in the cells. Using newer techniques of isotopic labeling and specific immune precipitation, Spiegelman and his colleagues were able to show that cells synthesize their enzymes de novo from amino acids, probably using some sort of molecular template or pattern which becomes activated by the inducer substance. Such a template, he noted, must be at least as large and complicated as the protein molecule which it is forming. And there were only two known possible candidates present in cells: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Various experiments by Spiegelman and other investigators demonstrated that enzyme synthesis in various microbes went on even when the DNA was damaged with radiation or the organism was grown in a medium lacking thymine. When RNA synthesis was impaired or stopped by various means, however, it almost always shut down enzyme synthesis. This suggested that the enzyme forming system in cells is a complex between RNA, inducer, and enzyme. From then on enzyme induction became one of the key systems used to understand gene function.