Concepts

In situ hybridization can be used to visualize the chromosomal location of a gene or to determine the tissue distribution of an mRNA transcribed from a specific gene. DNA footprinting is used to determine the sequences to which DNA-binding proteins attach.

Mutagenesis A powerful way to study gene function is to create mutations at specific locations in a process called site-directed mutagenesis and then to study the effects of these mutations on the organism.

A number of different strategies have been developed for site-directed mutagenesis. One strategy is to cut out a short sequence of nucleotides with restriction enzymes and replace it with a short, synthetic oligonulceotide that contains the desired mutated sequence (IFigure 18.21). The success of this method depends on the availability of restriction sites flanking the sequence to be altered.

If appropriate restriction sites are not available, oligonucleotide-directed mutagenesis can be used (I Figure 18.22). In this method, a single-stranded oligonucleotide is produced that differs from the target sequence by one or a few bases. Because they differ in only a few bases, the target DNA and the oligonucleotide will pair under the appropriate conditions. When successfully paired with the target DNA, the oligonucleotide can act as a primer to initiate DNA synthesis, which produces a double-stranded molecule with a mismatch in the primer region. When this DNA is transferred to bacterial cells, the mismatched bases will be repaired by bacterial enzymes. About half of the time the normal bases will be changed into mutant bases, and about half of the time the mutant bases will be changed into normal bases. The bacteria are then screened for the presence of the mutant gene.

A purified DNA fragment that is labeled at one end with 32P.

32p a a

.is bound tightly by a DNA-binding protein.

The DNA is cut at random locations .

Fragments are separated by gel electrophoresis.

No cleavage in area protected by DNA-binding protein

The DNA is cut at random locations .

Fragments are separated by gel electrophoresis.

^ The positions of the fragments with 32p on the gel are revealed by autoradiography.

No cleavage in area protected by DNA-binding protein

^ The positions of the fragments with 32p on the gel are revealed by autoradiography.

^ Because there is no cleavage in the area protected by the protein, a gap, or "footprint," appears in the ladder of bands, identifying bases that are bound by the protein.

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