In yeast, genes encoding transcription factors were first identified through classical genetic analysis. For example, one of the yeast genes required for growth on galactose is called GAL4. Incubation of wild-type yeast cells in galactose media results in more than a thousand-fold increase in the concentration of mRNAs encoding the enzymes catalyzing galactose metabolism. This activation of mRNA expression is not observed in gal4 mutants. (In S. cerevisiae, wild-type genes are designated with capital letters in italics, and recessive mutant alleles of the gene are indicated with lowercase letters in italics. The encoded protein is designated by the name of the gene in Roman type, with the first letter capitalized, e.g., Gal4.) Directed mutagenesis studies like those described previously identified UASs for the induced genes. Each of these UASs was found to contain one or more copies of a related 17-bp sequence called UASGAL. When a copy of UASGAL was cloned upstream of a TATA box followed by a lacZ reporter gene, expression of lacZ was activated in galactose media in wild-type cells, but not in gal4 mutants. This indicated that UASGAL is a transcription-control element activated by the Gal4 protein in galactose media.

The GAL4 gene was isolated by complementation of a gal4 mutant with a library of wild-type yeast DNA. By use of recombinant DNA techniques, the Gal4 protein was expressed in E. coli and found to bind to UASGAL. Thus, the Gal4 protein binds to UASGAL sequences and activates transcription from a nearby promoter when cells are placed in galactose media.

Classical genetic studies in a number of other organisms including Drosophila, the nematode C. elegans, and higher plants have uncovered several genes encoding transcription factors. For example, many mutations that interfere with normal Drosophila development have been identified. One of these inactivates the Ultrabithorax (Ubx) gene, causing an extra pair of wings to develop from the third thoracic segment. The protein encoded by wild-type Ubx has been shown to function as a transcription factor. The remarkable change in phenotype observed in Ubx mutants indicates that Ubx protein influences transcription of a large number of Drosophila genes.