polymerase this incubation period, RNA polymerase molecules that were actively transcribing genes when the nuclei were isolated incorporate several hundred labeled nucleotides into each nascent (growing) RNA chain, but little initiation of new chains occurs. The total radioactive label incorporated into RNA is a measure of the overall transcription rate. The fraction of the total labeled RNA produced by transcription of a particular gene—that is, its relative transcription rate—is determined by hybridizing the labeled RNA to the cloned DNA of that gene attached to a membrane.
The results of run-on transcription analyses illustrated in Figure 11-2 show that transcription of genes encoding proteins expressed specifically in hepatocytes (the major cell type in mammalian liver) is readily detected in nuclei pre-
pared from liver, but not in nuclei from brain or kidney. Since the run-on transcription assay measures RNA synthesis, these results indicate that differential synthesis of liver-specific proteins is regulated by controlling transcription of the corresponding genes in different tissues. Similar results have been obtained in run-on transcription experiments with other cell types and a wide variety of tissue-specific proteins, indicating that transcriptional control is the primary mechanism of gene control in complex organisms.
Regulatory Elements in Eukaryotic DNA Often Are Many Kilobases from Start Sites
In eukaryotes, as in bacteria, a DNA sequence that specifies where RNA polymerase binds and initiates transcription of a gene is called a promoter. Transcription from a particular promoter is controlled by DNA-binding proteins, termed transcription factors, that are equivalent to bacterial repressors and activators. However, the DNA control elements in eu-karyotic genomes that bind transcription factors often are located much farther from the promoter they regulate than is the case in prokaryotic genomes. In some cases, transcription factors that regulate expression of protein-coding genes in higher eukaryotes bind at regulatory sites tens of thousands of base pairs either upstream (opposite to the direction of transcription) or downstream (in the same direction as transcription) from the promoter. As a result of this arrangement, transcription from a single promoter may be regulated by binding of multiple transcription factors to alternative control elements, permitting complex control of gene expression.
For example, alternative transcription-control elements regulate expression of the mammalian gene that encodes transthyretin (TTR), which transports thyroid hormone in blood and the cerebrospinal fluid that surrounds the brain
M EXPERIMENTAL FIGURE 11-2 Measurement of RNA production in various tissues demonstrates that transcription of a given gene generally occurs only in the cell types in which it is expressed. Nuclei from mouse liver, kidney, and brain cells were exposed to 32P-UTR The three resulting labeled RNA samples were hybridized to separate nitrocellulose membranes; on each membrane was fixed an identical pattern of various cDNAs (top). The cDNAs labeled 1-12 (green) encode proteins synthesized actively in liver (e.g., albumin, transferrin) but not in most other tissues. The other cDNAs tested were actin (A) and a- and (3-tubulin (aT, pT) (yellow), which are proteins found in almost all cell types. A gene encoding methionine tRNA and the plasmid vector DNA (pV) in which the cDNAs were cloned were included as controls (purple). After removal of unhybridized RNAs, the labeled RNAs complementary to the cDNAs were revealed by autoradiography. RNA from the liver sample hybridized extensively with the cDNAs. Little hybridization is seen for the kidney and brain samples, indicating that genes encoding proteins found in hepatocytes are transcribed in liver cells but not in the cells of the other tissues. [See E. Derman et al., 1981, Cell 23:731, and D. J. Powell et al., 1984, J. Mol. Biol. 197:21.]
Was this article helpful?
Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.