Transfect each type of plasmid (1-5) separately into cultured cells

Reporter enzyme

Prepare cell extract and assay activity of reporter enzyme

Reporter-gene expression

Reporter plasmid

Reporter enzyme

▲ EXPERIMENTAL FIGURE 11-3 5'-Deletion analysis can identify transcription-control sequences in DNA upstream of a eukaryotic gene. Step 1 : Recombinant DNA techniques are used to prepare a series of DNA fragments that extend from the 5'-untranslated region of a gene various distances upstream. Step 2| : The DNA fragments are ligated into a reporter plasmid upstream of an easily assayed reporter gene. Step 3 : The DNA is transformed into E. coli to isolate plasmids with deletions of various lengths 5' to the transcription start site. Step 4|: Each plasmid is then transfected into cultured cells (or used to prepare transgenic organisms) and expression of the reporter gene is assayed (step 5 ). The results of this hypothetical example (bottom ) indicate that the test fragment contains two control elements. The 5' end of one lies between deletions 2 and 3; the 5' end of the other lies between deletions 4 and 5.

and spinal cord. Transthyretin is expressed in hepatocytes, which synthesize and secrete most of the blood serum proteins, and in choroid plexus cells in the brain, which secrete cerebrospinal fluid and its constituent proteins. The control elements required for transcription of the TTR gene were identified by the procedure outlined in Figure 11-3. In this experimental approach, DNA fragments with varying extents of sequence upstream of a start site are cloned in front of a reporter gene in a bacterial plasmid using recombinant DNA techniques. Reporter genes express enzymes that are easily assayed in cell extracts. Commonly used reporter genes include the E. coli lacZ gene encoding ^-galactosidase; the firefly gene encoding luciferase, which converts energy from ATP hydrolysis into light; and the jellyfish gene encoding green fluorescent protein (GFP).

By constructing and analyzing a 5' -deletion series upstream of the TTR gene, researchers identified two control elements that stimulate reporter-gene expression in hepato-cytes, but not in other cell types. One region mapped between «2.01 and 1.85 kb upstream of the TTR gene start site; the other mapped between «200 base pairs upstream and the start site. Further studies demonstrated that alternative DNA sequences control TTR transcription in choroid plexus cells. Thus, alternative control elements regulate transcription of the TTR gene in two different cell types. We examine the basic molecular events underlying this type of eukaryotic transcriptional control in later sections.

Three Eukaryotic Polymerases Catalyze Formation of Different RNAs

The nuclei of all eukaryotic cells examined so far (e.g., vertebrate, Drosophila, yeast, and plant cells) contain three different RNA polymerases, designated I, II, and III. These enzymes are eluted at different salt concentrations during ion-exchange chromatography and also differ in their sensitivity to a-amanitin, a poisonous cyclic octapeptide produced by some mushrooms (Figure 11-4). Polymerase I is very insensitive to a-amanitin; polymerase II is very sensitive; and polymerase III has intermediate sensitivity.

Each eukaryotic RNA polymerase catalyzes transcription of genes encoding different classes of RNA. RNA polymerase I, located in the nucleolus, transcribes genes encoding precursor rRNA (pre-rRNA), which is processed into 28S, 5.8S, and 18S rRNAs. RNA polymerase III transcribes genes encoding tRNAs, 5S rRNA, and an array of small, stable RNAs, including one involved in RNA splicing (U6) and the RNA component of the signal-recognition particle (SRP) involved in directing nascent proteins to the endoplasmic retic-ulum (Chapter 16). RNA polymerase II transcribes all protein-coding genes; that is, it functions in production of mRNAs. RNA polymerase II also produces four of the five small nuclear RNAs that take part in RNA splicing.

Each of the three eukaryotic RNA polymerases is more complex than E. coli RNA polymerase, although their structures are similar (Figure 11-5). All three contain two large

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