An "instant gene bank" method for heterologous gene cloning: complementation of two Aspergillus nidulans mutants with Gaeumannomyces graminis DNA.
We present a novel technique for gene cloning by complementation of mutations in Aspergillus nidulans with DNA from a heterologous organism, Gaeumannomyces graminis. This technique bypasses the time-consuming and difficult construction of gene libraries, making it both rapid and simple. The method relies on recombination between a fungal replicating vector pHELP1 and linear G. graminis genomic DNA during co-transformation. We were able to complement two out of seven A. nidulans mutants tested and to rescue transforming DNA from both in Escherichia coli. Complementation of the A. nidulans argB mutation resulted from integration of 8-10 kb segments of G. graminis DNA into pHELP1. The complementation of the A. nidulans pyrG mutation resulted from a complex rearrangement. Complementing DNA was shown to originate from G. graminis, and was capable of retransforming the original mutants to give the expected phenotype.
Bowyer
P
P
Sainsbury Laboratory, Norwich Research Park, Colney, UK.
Osbourn
A E
AE
Daniels
M J
MJ
D001233:Aspergillus nidulans / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular / Q000379:methods*; D005658:Fungi / Q000235:genetics*; D015723:Gene Library; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009154:Mutation; D010935:Plant Diseases / Q000382:microbiology; D010957:Plasmids; D015183:Restriction Mapping; D014170:Transformation, Genetic