Chromatin immunoprecipitation can also be used to deduce the cis-regulatory sequences recognized by a given transcription regulator. Here, all the DNA sequences precipitated by the regulator are lined up (by computer) and features in common are tabulated to produce the spectrum of cis-regulatory sequences recognized by the protein (see Figure 7–9A). Chromatin immunoprecipitation is also used routinely to identify the positions along a genome that are bound by the various types of modified histones discussed in Chapter 4. In this case, antibodies specific to the particular histone modification are employed (see Figure 8–67). A variation of the technique can also be used to map positions of chromosomes that are in physical proximity (see Figure 4–48).
Ribosome Profiling Reveals Which mRNAs Are Being Translated in the Cell
In preceding sections, we discussed several ways that RNA levels in the cell can be monitored. But for mRNAs, this represents only one step in gene expression, and we are often more interested in the final level of the protein produced by the gene. As described in the first part of this chapter, mass-spectroscopy methods can be used to monitor the levels of all proteins in the cell, including modified forms of the proteins. However, if we want to understand how synthesis of proteins is controlled by the cell, we need to consider the translation step of gene expression.
An approach called ribosome profiling provides an instantaneous map of the position of ribosomes on each mRNA in the cell and thereby identifies those many other DNA fragments that comprise the rest of the genome mRNAs that are being actively translated. To accomplish this, total RNA from a cell line or tissue is exposed to RNAses under conditions where only those RNA sequences covered by ribosomes are spared. The protected RNAs are released from ribosomes, converted to DNA, and the nucleotide sequence of each is determined (Figure 8–68). When these sequences are mapped on the genome, the position of ribosomes across each mRNA species can be ascertained.
Ribosome profiling has revealed many cases where mRNAs are abundant but are not translated until the cell receives an external signal. It has also shown that many open reading frames (ORFs) that were too short to be annotated as genes are actively translated and probably encode functional, albeit very small, proteins (Figure 8–69). Finally, ribosome profiling has revealed the ways that cells rapidly and globally change their translation patterns in response to sudden changes in temperature, nutrient availability, or chemical stress.
We have seen that nucleic acid methodologies developed in the past 40 years have completely changed the way that cell and molecular biology is studied. But they have also had a profound effect on our day-to-day lives. Many human pharmaceuticals in routine use (insulin, human growth hormone, blood-clotting factors, and interferon, for example) are based on cloning human genes and expressing the encoded proteins in large amounts. As DNA sequencing continues to drop in cost, more and more individuals will elect to have their genome sequenced; this information can be used to predict susceptibility to diseases (often with the option of minimizing this possibility by appropriate behavior) or to predict the way an individual will respond to a given drug. The genomes of tumor cells from an individual can be sequenced to determine the best type of anticancer treatment. And mutations that cause or greatly increase the risk of disease continue to be identified at an unprecedented pace. Using the recombinant DNA technologies discussed in this chapter, these mutations can then be introduced into animals, such as mice, that can be studied in the laboratory. The resulting transgenic animals, which often mimic some of the phenotypic abnormalities associated with the condition in patients, can be used to explore the cellular and molecular basis of the disease and to screen for drugs that could potentially be used therapeutically in humans.
number of sequence reads