Frederick G D, Asch D K, Kinsey J A
Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical School, Kansas City 66103.
Mol Gen Genet. 1989 Jun;217(2-3):294-300. doi: 10.1007/BF02464896.
Specific in vitro-generated insertion, replacement, and deletion mutations have been integrated near the chromosomal locus of am (NADP-specific glutamate dehydrogenase) of Neurospora crassa. Two approaches have been successful. One approach used am+-containing vectors capable of integrating at any site in the genome. This technique was used to introduce a specific 700 bp insertion near the am locus and to replace chromosomal sequences near am 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 the am region. A second approach increased the efficiency by using vectors containing a truncated am 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 the am region.
特定的体外产生的插入、替换和缺失突变已整合到粗糙脉孢菌am（NADP特异性谷氨酸脱氢酶）的染色体位点附近。有两种方法取得了成功。一种方法使用能够整合到基因组任何位点的含am⁺载体。该技术用于在am位点附近引入特定的700 bp插入，并用地质粒DNA替换am附近的染色体序列。然而，效率很低，必须筛选许多转化体才能在异位插入中找到所需的改变，除非导入的DNA与am区域有大片同源区域。第二种方法通过使用含有截短am基因的载体提高了效率，这样原养型只能通过同源重组产生。相对于第一种方法，总体转化频率降低了，但很大一部分转化涉及am区域的特定改变。
