A cosmid for selecting genes by complementation in Aspergillus nidulans : Selection of the developmentally regulated yA locus

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Abstract
A 9,9-kilobase cloning vector, designated pKBY2, was constructed for isolating genes by complementation of mutations in A. nidulans. pKBY2 contains the bacteriophage .lambda. cos site, to permit in vitro assembly of phage particles; a bacterial origin of replication and genes for resistance to ampicillin and chloramphenicol, to permit propagation in Escherichia coli; the A. nidulans trpC+ gene, to permit selection in Aspergillus; and a unique BamHI restriction site, to permit insertion of DNA fragments produced by digestion with restriction endonucleases BamHI, BglII, MboI or Sau3A. This cosmid was used to form a quasirandom recombinant DNA library containing 35- to 40-kilobase DNA fragments from a wild-type strain of A. nidulans. DNA from this library transformed a yellow-spored (yA-) pabaA- trpC- Aspergillus strain (FGSC237) to trpC+ at frequencies of .apprx. 10 transformants/.mu.g of DNA. Three of .apprx. 1000 trpC+ pabaA- colonies obtained were putative yA+ transformants, becuse they produced wild-type (green) spores. DNA from each of the green-spored transformants contained pKBY2 sequences and DNA from 2 transformants transduced E. coli to ampicillin resistance following treatment in vitro with a .lambda. packaging extract. The cosmids recovered in E. coli had similar restriction patterns and both yielded trpC+ transformants of A. nidulans FGSC237, 85% of which produced green spores. Several lines of evidence indicate that the recovered cosmids contain a wild-type copy of the yA gene.