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Isolation and expression of the acetate-inducible isocitrate lyase gene (acu-3) from Neurospora crassa: evidence for a second constitutive isozyme

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Summary

Heterologous hybridisation of the Aspergillus nidulans structural gene for isocitrate lyase (acuD) to a lambda genomic library of Neurospora crassa identified a recombinant phage containing the hybridising sequence on an internal 9 kb EcoRI fragment. A restriction fragment length polymorphism (RFLP) enabled the fragment to be assigned to linkage group V (LG V), the location of the acetate-inducible isocitrate lyase, acu-3 of Neurospora. Functional ectopic complementation by co-transformation of an am −, acu − double mutant using independent plasmid clones, carrying the entire 9 kb EcoRI fragment (pICLG1) and the selectable marker am + (NADP-glutamate dehydrogenase), demonstrated that the clone contains the entire acetate-inducible transcription unit. However, Northern analysis revealed two species of mRNA, only one of which was inducible on acetate. Native polyacrylamide gel electrophoresis separated two iso-enzymic activities, again only one of which was acetate-inducible and deficient in acu-3 − mutants. Further hybridisation of the acu-3 gene probe to an electrophoretic karyotype of Neurospora crassa identified sequences in an additional linkage group as well as in LG V, as anticipated. The isozymes are therefore sequence-related.

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Authors and Affiliations

  1. Department of Microbiology, University of Reading, London Road, RG1 5AQ, Reading, UK

    L. D. S. Gainey & I. F. Connerton

  2. MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, OX1 3QU, Oxford, UK

    K. Kölble

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  1. L. D. S. Gainey
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  2. K. Kölble
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  3. I. F. Connerton
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Communicated by J. van den Hondel

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Gainey, L.D.S., Kölble, K. & Connerton, I.F. Isolation and expression of the acetate-inducible isocitrate lyase gene (acu-3) from Neurospora crassa: evidence for a second constitutive isozyme. Molec. Gen. Genet. 229, 253–260 (1991). https://doi.org/10.1007/BF00272163

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  • DOI: https://doi.org/10.1007/BF00272163

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