The reverse genetic analysis starts with a known molecule— a DNA sequence, an mRNA, or a protein—and then attempts to disrupt this molecule in order to assess the role of the normal gene product in the biology of the organism. There are several approaches to reverse genetics. One approach is to mutagenize the genome randomly but then home in on the gene of interest by mapping or allelism tests by complementation. A second way is to conduct targeted mutagenesis that highly favors the production of mutations in the gene of interest. This disease is the most common form of inherited mental retardation, occurring in close
A third way is to create phenocopies—effects comparable to mutant phenotypes—by treatment with agents that inter- fere with mRNA or with the activity of the final protein product. There are advantages to each of these approaches. Random mutagenesis is the easiest to carry out, but it requires time and effort to sift through all the mutations to find the small proportion that includes the gene of interest. Targeted mutagenesis is also labor-intensive, but once the targeted mutation is obtained, it is more straightforward to characterize. Creating phenocopies can be very efficient, but there are limits to the kinds of phenotypes that can be copied. We will consider examples of each of these approaches. A knowledge of restriction sites is also useful in the directed mutation of a transgene.
For example, a small deletion can be made by removing the fragment that is liberated by cutting at two restriction sites (Figure 16-16b). With a similar double cut, a fragment, or cassette, can be inserted at a single restriction cut to create a duplication or other modification (Figure 16-16c). Another approach is to erode enzymatically a cut end created by a restriction enzyme to create deletions of various lengths (Figure 16-16d). PCR also can be used to generate a DNA fragment containing a specific mutation, for eventual introduction as a transgene (Figure 16-16e). Base insertion and deletion Although some errors in replication produce base-substitution mutations, other kinds of replication errors can lead to indel mutations—that is, insertions or deletions of one or more base pairs.
When such mutations add or subtract a number of bases not divisible by three, they produce frameshift mutations in the protein-coding regions. The nucleotide sequence at frameshift mutation hot spots was determined in the lysozyme-encoding gene of phage T4. These mutations often occur at repeated bases. The prevailing model (Figure 14-21) proposes that indels arise when loops in single-stranded regions are stabilized by the “slipped mispairing” of repeated sequences in the course of replication. This mechanism is sometimes called replication slippage. In the E. coli lacI gene, certain hot spots result from repeated sequences, just as predicted by this model. Figure 14-22 depicts the distribution of spontaneous mutations in the lacI gene.
Note how one site dominates the distribution. In lacI, the major indel hot spot is a four-base pair sequence (CTGG) repeated three times in tandem in the wild type (for simplicity, only one strand of the DNA is shown): The majority of mutations at this site (represented here by the mutations FS5, FS25, FS45, and FS65) result from the addition of one extra set of the four bases CTGG. A minority (represented here by the mutations FS2 and FS84) results from the loss of one set of the four bases CTGG. How can we explain these observations? The model predicts that the frequency of a particular indel depends on the number of base pairs that can form during the slipped mispairing of repeated sequences.
The wild-type sequence shown for the lacI gene can slip out one CTGG sequence and stabilize this structure by forming nine base pairs (apply the model in Figure 14-21 to the sequence shown for lacI). Whether a deletion or an insertion is generated depends on whether the slippage is on the template or on the newly synthesized strand, respectively. Larger deletions (more than a few base pairs) constitute a sizable fraction of observed spontaneous mutations, as shown in Figure 14-22. Most, although not all of the deletions are of repeated sequences. Figure 14-23 shows 9 deletions analyzed at the DNA sequence level in the lacI gene of E. coli. The results of further studies have shown that longer repeats constitute hot spots for deletions.
Duplications of DNA segments have also been observed in many organisms. Like deletions, they often occur at sequence repeats. It must be noted that, in addition to their origin by replication slippage, deletions and duplications could be generated by offset homologous recombination between copies of the repeats. Spontaneous mutations in humans— trinucleotide repeat diseases DNA sequence analysis has revealed the gene mutations contributing to numerous human hereditary diseases. Many are of the expected base-substitution or single base-pair indel type. However, some mutations are more complex. A number of these human disorders are due to duplications of short repeated sequences. A common mechanism responsible for a number of genetic diseases is the expansion of a three-base-pair repeat. For this reason, they have termed trinucleotide repeat diseases.
A third way is to create phenocopies—effects comparable to mutant phenotypes—by treatment with agents that inter- fere with mRNA or with the activity of the final protein product. There are advantages to each of these approaches. Random mutagenesis is the easiest to carry out, but it requires time and effort to sift through all the mutations to find the small proportion that includes the gene of interest. Targeted mutagenesis is also labor-intensive, but once the targeted mutation is obtained, it is more straightforward to characterize. Creating phenocopies can be very efficient, but there are limits to the kinds of phenotypes that can be copied. We will consider examples of each of these approaches. A knowledge of restriction sites is also useful in the directed mutation of a transgene.
For example, a small deletion can be made by removing the fragment that is liberated by cutting at two restriction sites (Figure 16-16b). With a similar double cut, a fragment, or cassette, can be inserted at a single restriction cut to create a duplication or other modification (Figure 16-16c). Another approach is to erode enzymatically a cut end created by a restriction enzyme to create deletions of various lengths (Figure 16-16d). PCR also can be used to generate a DNA fragment containing a specific mutation, for eventual introduction as a transgene (Figure 16-16e). Base insertion and deletion Although some errors in replication produce base-substitution mutations, other kinds of replication errors can lead to indel mutations—that is, insertions or deletions of one or more base pairs.
When such mutations add or subtract a number of bases not divisible by three, they produce frameshift mutations in the protein-coding regions. The nucleotide sequence at frameshift mutation hot spots was determined in the lysozyme-encoding gene of phage T4. These mutations often occur at repeated bases. The prevailing model (Figure 14-21) proposes that indels arise when loops in single-stranded regions are stabilized by the “slipped mispairing” of repeated sequences in the course of replication. This mechanism is sometimes called replication slippage. In the E. coli lacI gene, certain hot spots result from repeated sequences, just as predicted by this model. Figure 14-22 depicts the distribution of spontaneous mutations in the lacI gene.
Note how one site dominates the distribution. In lacI, the major indel hot spot is a four-base pair sequence (CTGG) repeated three times in tandem in the wild type (for simplicity, only one strand of the DNA is shown): The majority of mutations at this site (represented here by the mutations FS5, FS25, FS45, and FS65) result from the addition of one extra set of the four bases CTGG. A minority (represented here by the mutations FS2 and FS84) results from the loss of one set of the four bases CTGG. How can we explain these observations? The model predicts that the frequency of a particular indel depends on the number of base pairs that can form during the slipped mispairing of repeated sequences.
The wild-type sequence shown for the lacI gene can slip out one CTGG sequence and stabilize this structure by forming nine base pairs (apply the model in Figure 14-21 to the sequence shown for lacI). Whether a deletion or an insertion is generated depends on whether the slippage is on the template or on the newly synthesized strand, respectively. Larger deletions (more than a few base pairs) constitute a sizable fraction of observed spontaneous mutations, as shown in Figure 14-22. Most, although not all of the deletions are of repeated sequences. Figure 14-23 shows 9 deletions analyzed at the DNA sequence level in the lacI gene of E. coli. The results of further studies have shown that longer repeats constitute hot spots for deletions.
Duplications of DNA segments have also been observed in many organisms. Like deletions, they often occur at sequence repeats. It must be noted that, in addition to their origin by replication slippage, deletions and duplications could be generated by offset homologous recombination between copies of the repeats. Spontaneous mutations in humans— trinucleotide repeat diseases DNA sequence analysis has revealed the gene mutations contributing to numerous human hereditary diseases. Many are of the expected base-substitution or single base-pair indel type. However, some mutations are more complex. A number of these human disorders are due to duplications of short repeated sequences. A common mechanism responsible for a number of genetic diseases is the expansion of a three-base-pair repeat. For this reason, they have termed trinucleotide repeat diseases.
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