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. 1977 Aug;74(8):3508–3512. doi: 10.1073/pnas.74.8.3508

Expression in Escherichia coli K-12 of the structural gene for catabolic dehydroquinase of Neurospora crassa

Daniel Vapnek *, Judith A Hautala †, James W Jacobson †, Norman H Giles †, Sidney R Kushner ‡
PMCID: PMC431620  PMID: 143663

Abstract

The inducible quinic acid catabolic pathway of Neurospora crassa is controlled by four genes, the qa cluster which includes structural genes qa-2, qa-3, qa-4 for three enzymes and a regulatory gene, qa-1. In this paper we report the molecular cloning of at least the qa-2 gene which encodes the catabolic dehydroquinase (5-dehydroquinate hydro-lyase, EC 4.2.1.10). Endo·R·HindIII restriction endonuclease fragments of N. crassa DNA from a qa-1c (constitutive) mutant and of Escherichia coli plasmid pBR322 DNA were ligated in vitro and used to transform an aroD6 (5-dehydroquinate hydrolyase deficient) strain of E. coli K12. The recombinant plasmid (pVK55) isolated from one AroD+ transformant (SK1518) contained, in addition to pBR322, two N. crassa HindIII fragments with molecular weights of 2.3 × 106 and 1.9 × 106. Derivatives of SK1518 cured of plasmid DNA were phenotypically Amps and AroD-. These cured strains, retransformed with pVK55, were phenotypically AmpR and AroD+. Strains transformed with pVK55 possessed 5-dehydroquinate hydrolyase activity but no activity was present in any AroD- strain. The enzyme extracted from strains containing the recombinant plasmid was identical to N. crassa catabolic dehydroquinase by the criteria of heat stability, ammonium sulfate fractionation, immunological crossreactivity, molecular weight, and purification characteristics. This identity demonstrates that the N. crassa qa-2+ gene is carried by the recombinant plasmid and is apparently transcribed and translated with complete fidelity. Furthermore, subunit assembly of the N. crassa polypeptides also occurs in E. coli, because the catabolic dehydroquinase is a multimer composed of approximately 20 identical subunits.

Keywords: recombinant DNA, pBR322 plasmid, eukaryotic gene regulation, qa cluster

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Selected References

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