Use of transformation to make targeted sequence alterations at theam (GDH) locus ofNeurospora
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Specific in vitro-generated insertion, replacement, and deletion mutations have been integrated near the chromosomal locus ofam (NADP-specific glutamate dehydrogenase) ofNeurospora crassa. Two approaches have been successful. One approach usedam +-containing vectors capable of integrating at any site in the genome. This technique was used to introduce a specific 700 bp insertion near theam locus and to replace chromosomal sequences nearam with plasmid DNA. Efficiency was low, however, and many transformants had to be screened to find the desired alterations among the ectopic insertions unless the incoming DNA had a large region of homology with theam region. A second approach increased the efficiency by using vectors containing a truncatedam gene, so that prototrophs could arise only by homologous recombination. Overall transformation frequency was reduced relative to the first method, but a large fraction of the transformations involved specific alterations of theam region.
Key wordsIn vitro mutagenesis Gene replacement Deletions Insertions
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