Altering Cloned DNA by in vitro Mutagenesis
Understanding DNA-related biological mechanisms requires more than characterizing the DNA and associated proteins. It often requires altera-tion of the components. Not only does variation of the relevant parame-ters reveal more about the working mechanism, but the ability to test variants permits definitive proof of theories. Mutants have been used in molecular biology almost from its origins, first in the elucidation of biochemical pathways and now prominently in structural studies of the mechanisms by which proteins function as enzymes or recognize and bind to specific nucleotide sequences on DNA.
The efficient isolation of mutations has always posed a problem in molecular biology. Suppose mutations are desired in a particular gene or DNA sequence. If the entire organism must be mutagenized, then to obtain a reasonable number of alterations in the desired target, many more alterations will inevitably occur elsewhere on the chromosome. Often these other mutations will be lethal, so the necessary alterations in the target cannot easily be found. A method is needed for directing mutations just to the target gene. In vitro mutagenesis of cloned DNA fragments is a solution to the problem. Only the DNA of the target sequence is mutagenized. Just this sequence is then put back into cells.
Often random mutations need to be directed to small areas of genes or to specific nucleotides, or specific changes are desired in specific nucleotides. Some changes are easy to make. For example, insertions and deletions can be generated at the cleavage site of a restriction enzyme. A four-base insertion can be generated at the cleavage site of BamHI by filling in the four-base single-stranded ends with DNA pol Iand ligating the flush ends together.
Similarly, a four-base deletion can be generated by digesting the single-stranded ends with the single-stranded specific nuclease S1 be-fore ligation. Variations on these themes are to use DNA pol I in the presence of only one, two, or three of the nucleotides to fill out part of the single-stranded ends before nuclease treatment and ligation (Fig. 10.20). Mixing and matching entire restriction fragments from a region under study is another closely related method of changing portions of DNA binding sites or substituting one portion of a protein for another.
More extensive deletions from the ends of DNA molecules can be generated by double-stranded exonuclease digestion. The nuclease Bal 31 from the culture medium of the bacterium Alteromonas espejiana is particularly useful for this purpose. With it, a set of clones with progres-sively larger deletions into a region can easily be isolated. The addition of linkers after Bal 31 digestion permits targeted substitution of a set of nucleotides or a change in the number of nucleotides between two sites. Deletions entering the region from both directions are isolated. Before recloning, a restriction enzyme linker is added. After these steps, a pair of deletions can be easily joined via their linkers to generate a DNA molecule identical to the wild-type except for the alteration of a stretch comprising the linker (Fig. 10.21). The use of different pairs of deletions place the linker in different locations so that the linker can be scanned through a region to determine important areas.
Bases within DNA fragments can be changed with chemical in vitro mutagenesis. Hydroxylamine will effectively mutagenize the cytosines in denatured DNA fragments, which can then be renatured and re-cloned.
Figure 10.21 Digestion with Bal 31 from either direction and addition oflinkers generates a set of molecules that can be rejoined via the linkers to yield a molecule like the original wild type but with a substitution of some nucleo-tides.
Alternatively, mutagenesis can be directed to particular regions. One method is to generate a single-stranded region by nicking one strand as a result of digestion with a restriction enzyme in the presence of ethidium bromide and then briefly digesting with exonuclease III to generate a gap. The mutagenesis is then performed with a single-stranded specific reagent such as sodium bisulfite, which mutagenizes cytosines and ultimately converts them to thymines, or by compelling misincorporation of bases during repair of a gap.
Figure 10.22 Insertional inactivation of a yeast gene.
Insertional inactivation can be used to kill specific genes in yeast (Fig. 10.22). This is a prerequisite to examining the in vivo consequences of mutating the gene. Suppose that a cloned copy of the gene to be inactivated is available. Then the central portion of the gene can be replaced by a segment of DNA encoding one of the genes necessary for the synthesis of uracil. Uracil-requiring yeast cells are transformed with the segment of DNA containing the gene segments and the URA region, and selection is performed for cells able to grow without exogenously added uracil. Since the ends of the transforming DNA segment are highly recombinogenic, the fragment recombines into the X gene with high frequency and replaces the former intact copy of the X gene with the damaged copy. This replacement relieves the uracil requirement of the cells. That the necessary construct has been generated can be verified by Southern transfers. Restriction sites flanking the insertion are moved further apart, increasing the size of this restriction fragment.
When the steps described above are performed on diploid yeast cells, the result is one chromosome with an insertionally inactivated copy of the X gene and a second, normal copy of the X gene. To test whether the X gene is required for growth in haploid cells, haploids containing the two chromosome types can then be generated by sporulating the diploids. If the gene with the insertion is completely inviable, only two of the four spores from each tetrad will be viable.
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