EXPERIMENTAL FIGURE 9-22 Linker scanning mutations identify transcription-control elements. (a) In linker scanning mutagenesis, a region of eukaryotic DNA (tan) that supports high-level expression of a reporter gene (light purple) is cloned in a plasmid vector as diagrammed at the top. Overlapping linker scanning (LS) mutations (crosshatched areas) are introduced from one end of the region being analyzed to the other. These mutations are created by scrambling the nucleotide sequence in a short stretch of the DNA. After the mutant plasmids are transfected separately into cultured cells, the activity of the reporter-gene product is assayed. In the example shown here, the sequence from −120 to +1 of the herpes simplex virus thymidine kinase gene, LS mutations 1, 4, 6, 7, and 9 have little or no effect on expression of the reporter gene, indicating that the regions altered in these mutants contain no control elements. Reporter-gene expression is significantly reduced in mutants 2, 3, 5, and 8, indicating that control elements (brown) lie in the intervals shown at the bottom. (b) Analysis of these LS mutations identified a TATA box and two promoter-proximal elements (PE-1 and PE-2). See S. L. McKnight and R. Kingsbury, 1982, Science 217:316.