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Mild RIP — an alternative method for in vivo mutagenesis of thealbino-3 gene inNeurospora crassa

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Abstract

We have used a biological phenomenon that occurs inNeurospora crassa, termed Repeat-Induced Point mutation (RIP), to create partially functional mutant alleles of thealbino-3 (al-3) gene encoding geranylgeranyl pyrophosphate synthase, an enzyme involved in the biosynthesis of carotenoids and diverse prenylated compounds. A total of 70 RIP-inducedal- 3 mutants were identified by their pale albino phenotype, resulting from inactivation of carotenoid biosynthesis. Nucleotide sequence analysis of theal-3 gene in five of the RIP-induced mutants revealed that in each case RIP had introduced no more than six point mutations. The low frequency of RIP mutants (0.42%) and the isolation of only leaky mutants with very few mutations suggest that ascospores containing a heavily mutatedal-3 gene do not survive. These results are evidence that the RIP phenomenon, used to inactivate and silence duplicated genes inN. crassa, may be exploited in its mild version as a method of sequence-specific in vivo mutagenesis to obtain functional mutant alleles ofNeurospora genes. This mild form of mutagenesis may be particularly advantageous in selecting for leaky mutations in essentialNeurospora genes.

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Authors and Affiliations

  1. Dipartimento di Biopatologia Umana, Sezione Biologia Cellulare, Policlinico Umberto I, Università di Roma «La Sapienza», Viale Regina Elena, 324, 1-00161, Rome, Italy

    C. Barbato, M. Calissano, A. Pickford, N. Romano & G. Macino

  2. Biosynthesis Group, Botanische Institut, Postfach 11 19 32, D-60054, Frankfurt, Germany

    G. Sandmann

Authors
  1. C. Barbato
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  2. M. Calissano
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  3. A. Pickford
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  4. N. Romano
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  5. G. Macino
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  6. G. Sandmann
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Communicated by E. Cerdá-Olmedo

C.B. and M.C. contributed equally to this work

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Barbato, C., Calissano, M., Pickford, A. et al. Mild RIP — an alternative method for in vivo mutagenesis of thealbino-3 gene inNeurospora crassa . Molec. Gen. Genet. 252, 353–361 (1996). https://doi.org/10.1007/BF02172999

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  • DOI: https://doi.org/10.1007/BF02172999

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