AN (INSTANT GENE BANK) METHOD FOR GENE CLONING BY MUTANT COMPLEMENTATION

We describe a new method of gene cloning by complementation of mutant alleles which obviates the need for construction of a gene library in a plasmid vector in vitro and its amplification in Escherichia coli. T he method involves simultaneous transformation of mutant strains of th e fungus Aspergillus nidulans with (i) fragmented chromosomal DNA from a donor species and (ii) DNA of a plasmid without a selectable marker gene, but with a fungal origin of DNA replication ('helper plasmid'). Transformant colonies appear as the result of the joining of chromoso mal DNA fragments carrying the wild-type copies of the mutant allele w ith the helper plasmid. Joining may occur either by ligation (if the h elper plasmid is in linear form) or recombination (if it is cccDNA). T his event occurs with high efficiency in vivo, and generates an autono mously replicating plasmid cointegrate. Transformants containing Penic illium chrysogenum genomic DNA complementing A. nidulans niaD, nirA an d argB mutations have been obtained. While some of these cointegrates were evidently rearranged or consisted only of unaltered replicating p lasmid, in other cases plasmids could be recovered into E. coli and we re subsequently shown to contain the selected gene. The utility of thi s ''instant gene bank'' technique is demonstrated here by the molecula r cloning of the P. canescens trpC gene.