Advertisement

Molecular and General Genetics MGG

, Volume 262, Issue 6, pp 1022–1035 | Cite as

An osmotic-remedial, temperature-sensitive mutation in the allosteric activity site of ribonucleotide reductase in Neurospora crassa

  • M. L. Smith
  • S. P. Hubbard
  • D. J. Jacobson
  • O. C. Micali
  • N. L. Glass
ORIGINAL PAPER

Abstract

An osmotic-remedial, temperature-sensitive conditional mutant (un-24) was generated by Repeat Induced Point mutation (RIP) from a cross between a wild-type N. crassa strain and a strain carrying a ≈250-kb duplication of the left arm of linkage group II (LGII). The mutation was mapped to the duplicated segment, within 2.6 map units of the heterokaryon incompatibility locus het-6. DNA transformation identified a 3.75-kb fragment that complemented the temperature-sensitive phenotype. A large ORF within this fragment was found to have a high degree of sequence identity to the large subunit of ribonucleotide reductase (RNR) from diverse organisms. Conserved amino acids at the active site and the allosteric activity sites are also evident. An unusual feature of the Neurospora sequence is a large insertion near the C-terminus relative to otherwise homologous sequences from other organisms. Three transition mutations, indicative of RIP, were identified in the N-terminal region of the temperature-sensitive mutant allele. One of these mutations results in a non-conservative amino acid substitution within the four-helix bundle that is important in the allosteric control of ribonucleotide reductase activity. This substitution appears to disrupt proper folding of the allosteric activity site during synthesis of the protein.

Key wordsN. crassa Osmotic-remedial temperature-sensitive mutation Ribonucleotide reductase Repeat-induced point mutation (RIP) Hydroxyurea 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Copyright information

© Springer-Verlag Berlin Heidelberg 2000

Authors and Affiliations

  • M. L. Smith
    • 1
  • S. P. Hubbard
    • 1
  • D. J. Jacobson
    • 2
  • O. C. Micali
    • 1
  • N. L. Glass
    • 3
  1. 1.Biology Department, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario K1S 5B6, Canada E-mail: mysmith@ccs.carleton.ca Tel.: +1-613-5203864; Fax: +613-520-4497CA
  2. 2.Department of Biology, Stanford University, Stanford, CA 94305, USAUS
  3. 3.Department of Botany and The Biotechnology Laboratory, University of British Columbia, Vancouver, B.C. V6T 1Z3, CanadaCA

Personalised recommendations