Figure 8–67 Results of several chromatin immunoprecipitations showing proteins bound to the control region that control expression of the Oct4 gene. In this series of chromatin immunoprecipitation experiments, antibodies directed against a transcription regulator (first three panels) or a particular histone modification (fourth panel) were used to precipitate bound, cross-linked DNA. Precipitated DNA was sequenced, and the positions across the genome were mapped. (Only the small part of the mouse genome containing the Oct4 gene is shown.) The results show that, in the embryonic stem cells analyzed in these experiments, Oct4 binds upstream of its own gene and that Sox2 and Nanog are bound in close proximity. Oct4, Sox2, and Nanog are key regulators in embryonic stem cells (discussed in Chapter 22) and this experiment reveals the position on the genome through which they exert their effects on Oct4 expression. In the fourth panel, the positions of a histone modification associated with actively transcribed genes is shown (see Figure 4–39). Finally, the bottom panel shows the RNA produced from the Oct4 gene under the same conditions used for the chromatin immunoprecipitations. Note that the introns and exons are relatively easy to identify from these RNA-seq data.
Figure 8–68 Ribosome profiling. RNA
AUG UGA AAAAAAAAAAAA is purified from cells and digested with an RNAse to leave only those portions AUG of the mRNAs that are protected by a bound ribosome. These short pieces remove ribosomes convert RNA to DNA and sequence
Although we tend to think of recombinant DNA research in terms of animal biology, these techniques have also had a profound impact on the study of plants. In fact, certain features of plants make them especially amenable to recombinant DNA methods.
When a piece of plant tissue is cultured in a sterile medium containing nutrients and appropriate growth regulators, some of the cells are stimulated to proliferate indefinitely in a disorganized manner, producing a mass of relatively undifferentiated cells called a callus. If the nutrients and growth regulators are carefully manipulated, one can induce the formation of a shoot within the callus, and in many species a whole new plant can be regenerated from such shoots. In a number of plants—including tobacco, petunia, carrot, potato, and Arabidopsis—a single cell from such a callus (known as a totipotent cell) can be grown into a small clump of cells from which a whole plant can be regenerated (see Figure 7–2B). Just as mutant mice can be derived by the genetic manipulation of embryonic stem 200 ORF discovered through ribosome profling nucleotide pairs codes for a protein of 20 amino acids of protected RNA (approximately 20 nucleotides in length) are converted to DNA and sequenced. The resulting information is displayed as the number of sequence reads along each position of the genome. In the diagram here, the data for only one gene, whose mRNA is being efficiently translated, are shown. Ribosome profiling provides this type of information for every mRNA produced by the cell.
Figure 8–69 Ribosome profiling can identify new genes. This experiment shows the discovery of a previously unrecognized gene—one that encodes a protein of only 20 amino acids. At the top is shown a portion of a viral genome with two previously annotated genes. Below are the results of a ribosome profiling experiment, displayed across the same section of the genome, after the virus was infected into human cells. The results show that the left-hand gene is not expressed under these conditions, the right-hand gene is expressed at low levels, and a previously unrecognized gene that lies between them position along genome is expressed at high levels.
508 Chapter 8: Analyzing Cells, Molecules, and Systems leaf discs incubated with genetically engineereddiscs removed from cells in culture, so transgenic plants can be created from plant cells transfected with DNA in culture (Figure 8–70).