G

Syn*hetic U G G G A A G A C AUG UGG U U C G U probes G U U

20 nucleotides long, 8 possible sequences

FIGURE 9-10 Probe to detect the gene for a protein of known amino acid sequence. Because more than one DNA sequence can code for any given amino acid sequence, the genetic code is said to be "degenerate." (As described in Chapter 27, an amino acid is coded for by a set of three nucleotides called a codon. Most amino acids have two or more codons; see Fig. 27-7.) Thus the correct DNA sequence for a known amino acid sequence cannot be known in advance. The probe is designed to be complementary to a region of the gene with minimal degeneracy, that is, a region with the fewest possible codons for the amino acids—two codons at most in the example shown here. Oligonucleotides are synthesized with selectively randomized sequences, so that they contain either of the two possible nucleotides at each position of potential degeneracy (shaded in pink). The oligonucleotide shown here represents a mixture of eight different sequences: one of the eight will complement the gene perfectly, and all eight will match at least 17 of the 20 positions.

Bacterial promoter (P) and operator (O) sequences

Bacterial promoter (P) and operator (O) sequences

resistance)

FIGURE 9-11 DNA sequences in a typical E. coli expression vector.

The gene to be expressed is inserted into one of the restriction sites in the polylinker, near the promoter (P), with the end encoding the amino terminus proximal to the promoter. The promoter allows efficient transcription of the inserted gene, and the transcription termination sequence sometimes improves the amount and stability of the mRNA produced. The operator (O) permits regulation by means of a repressor that binds to it (Chapter 28). The ribosome binding site provides sequence signals needed for efficient translation of the mRNA derived from the gene. The selectable marker allows the selection of cells containing the recombinant DNA.

resistance)

FIGURE 9-11 DNA sequences in a typical E. coli expression vector.

The gene to be expressed is inserted into one of the restriction sites in the polylinker, near the promoter (P), with the end encoding the amino terminus proximal to the promoter. The promoter allows efficient transcription of the inserted gene, and the transcription termination sequence sometimes improves the amount and stability of the mRNA produced. The operator (O) permits regulation by means of a repressor that binds to it (Chapter 28). The ribosome binding site provides sequence signals needed for efficient translation of the mRNA derived from the gene. The selectable marker allows the selection of cells containing the recombinant DNA.

Alterations in Cloned Genes Produce Modified Proteins

Cloning techniques can be used not only to overproduce proteins but to produce protein products subtly altered from their native forms. Specific amino acids may be replaced individually by site-directed mutagenesis.

This powerful approach to studying protein structure and function changes the amino acid sequence of a protein by altering the DNA sequence of the cloned gene. If appropriate restriction sites flank the sequence to be altered, researchers can simply remove a DNA segment and replace it with a synthetic one that is identical to the original except for the desired change (Fig. 9-12a). When suitably located restriction sites are not present, an approach called oligonucleotide-directed muta-genesis (Fig. 9-12b) can create a specific DNA sequence change. A short synthetic DNA strand with a specific base change is annealed to a single-stranded copy of the cloned gene within a suitable vector. The mismatch of a single base pair in 15 to 20 bp does not prevent annealing if it is done at an appropriate temperature. The annealed strand serves as a primer for the synthesis of a strand complementary to the plasmid vector. This slightly mismatched duplex recombinant plasmid is then used to transform bacteria, where the mismatch is repaired by cellular DNA repair enzymes (Chapter 25). About half of the repair events will remove and replace the altered base and restore the gene to its original sequence; the other half will remove and

restriction endonucleases

Recombinant plasmid DNA

Gene

Gene

Single strand of recombinant plasmid DNA

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