H. Shiotani et T. Tsuge, EFFICIENT GENE TARGETING IN THE FILAMENTOUS FUNGUS ALTERNARIA-ALTERNATA, MGG. Molecular & general genetics, 248(2), 1995, pp. 142-150
To characterize homologous recombination of transforming DNA in the fi
lamentous fungus Alternaria alternata, we have compared the frequencie
s of gene targeting by circular and linear DNA fragments in the fungus
. The A. alternata BRM1 gene, which is an essential gene for melanin b
iosynthesis, was selected as a target locus. BRM1 targeting events are
easily identified because loss of function leads to a change in mycel
ial color from black to light brown. We constructed targeting vectors
by inserting 0.6 to 3.1 kb internal BRM1 segments into a plasmid conta
ining the hygromycin B phosphotransferase gene. When circular plasmids
were used, melanin-deficient (Me1(-)) transformants accounted for 30
to 80% of hygromycin B-resistant (Hy(R)) transformants, correlating cl
osely with the size of the BRM1 segment in the transforming DNA. Restr
iction enzyme digestion within the BRM1 region greatly enhanced the fr
equency of gene targeting: integration of the linear plasmids was almo
st completely attributable to homologous recombination, regardless of
the size of the BRM1 segments. Plasmids carrying both BRM1 segments an
d rDNA segments were transformed into the fungus to examine the effect
of the number of target copies on homologous recombination. Using the
circular plasmids, Me1(-) transformants accounted for only 5% of Hy(R
) transformants. In contrast, when the linear plasmid produced by rest
riction enzyme digestion within the BRM1 segment was used, almost all
transformants were Me1(-). These results indicate that homologous inte
gration of circular molecules in A. alternata is sensitive to the leng
th of homology and the number of targets, and that double-strand break
s in transforming DNA. greatly enhance homologous recombination.