An ‘instant gene bank’ method for gene cloning by mutant complementation

Abstract

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. The method involves simultaneous transformation of mutant strains of the 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 chromosomal DNA fragments carrying the wild-type copies of the mutant allele with the helper plasmid. Joining may occur either by ligation (if the helper plasmid is in linear form) or recombination (if it is cccDNA). This event occurs with high efficiency in vivo, and generates an autonomously replicating plasmid cointegrate. Transformants containing Penicillium chrysogenum genomic DNA complementing A. nidulans niaD, nirA and argB mutations have been obtained. While some of these cointegrates were evidently rearranged or consisted only of unaltered replicating plasmid, in other cases plasmids could be recovered into E. coli and were subsequently shown to contain the selected gene. The utility of this "instant gene bank" technique is demonstrated here by the molecular cloning of the P. canescens trpC gene.

The Aspergillus nidulans cysA gene encodes a novel type of serine O-acetyltransferase which is homologous to homoserine O-acetyltransferases The GenBank accession number for the sequence reported in this paper is AF029885.

“…To identify the region of the cloned cosmid complementing the mutation we used the ' instant gene bank ' method of Gems et al (1994). The method is based on the observation that cotransformation of a strain with the restricted pHELP1 plasmid and transforming DNA leads to in vivo ligation of DNA fragments with the vector.…”

Cloning Heterologous Genes: Problems and Approaches

“…Furthermore, shuttle cloning vectors that function and replicate in both Escherichia coli and the host organism are highly desirable for transformation and recovery of the gene. Such vectors are available for S. cerevisiae (Ausubel et al, 1995;Brunelli and Pall, 1993;Parent et al, 1985;Silar and Thiele, 1991), S. pombe (Brun et al, 1995;Cottarel et al, 1993), and Aspergillus species (Bowyer et al, 1994;Gems et al, 1994). No such vectors are available for N. crassa, where the much slower process of sib-selection, requiring several rounds of transformation, is still the only available screening method.…”

Expression of pigmentation genes following electroporation of albino Monascus purpureus

“…Genomic DNA uptake and expression has been demonstrated in Trichoderma harzianum and Gliocladium virens, where both biolistic and protoplast-mediated methods were used with fungal genomic DNA carrying a hygromycin resistance gene as the transforming DNA [14]. To identify fungal genes, an ''instant gene bank method'' in which a mutant recipient strain is transformed with genomic DNA from a wild-type strain, along with a helper plasmid carrying a fungal origin of replication, was proposed [2,9]. A major advantage of this approach is that it bypasses the need for the construction of gene libraries [2,9], which is a time-consuming and difficult process [2].…”

“…To identify fungal genes, an ''instant gene bank method'' in which a mutant recipient strain is transformed with genomic DNA from a wild-type strain, along with a helper plasmid carrying a fungal origin of replication, was proposed [2,9]. A major advantage of this approach is that it bypasses the need for the construction of gene libraries [2,9], which is a time-consuming and difficult process [2]. However, the method relies on recombination between the helper plasmid and linear wild-type DNA during co-transformation, and the resulting transformants were frequently mitotically unstable [2,9].…”