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Genetics, Vol 142, 117-127, Copyright © 1996
INVESTIGATIONS |
A UV-Induced Mutation in Neurospora That Affects Translational Regulation in Response to Arginine
M. Freitag, N. Dighde and M. S. Sachs
Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science & Technology, Portland, Oregon 97291-1000
The Neurospora crassa arg-2 gene encodes the small subunit of arginine-specific carbamoyl phosphate synthetase. The levels of arg-2 mRNA and mRNA translation are negatively regulated by arginine. An upstream open reading frame (uORF) in the transcript's 5' region has been implicated in arginine-specific control. An arg-2-hph fusion gene encoding hygromycin phosphotransferase conferred arginine-regulated resistance to hygromycin when introduced into N. crassa. We used an arg-2-hph strain to select for UV-induced mutants that grew in the presence of hygromycin and arginine, and we isolated 46 mutants that had either of two phenotypes. One phenotype indicated altered expression of both arg-2-hph and arg-2 genes; the other, altered expression of arg-2-hph but not arg-2. One of the latter mutations, which was genetically closely linked to arg-2-hph, was recovered from the 5' region of the arg-2-hph gene using PCR. Sequence analyses and transformation experiments revealed a mutation at uORF codon 12 (Asp to Asn) that abrogated negative regulation. Examination of the distribution of ribosomes on arg-2-hph transcripts showed that loss of regulation had a translational component, indicating the uORF sequence was important for Arg-specific translational control. Comparisons with other uORFs suggest common elements in translational control mechanisms.
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Z. Wang and M. S. Sachs Arginine-specific Regulation Mediated by the Neurospora crassa arg-2 Upstream Open Reading Frame in a Homologous, Cell-free in Vitro Translation System J. Biol. Chem., January 3, 1997; 272(1): 255 - 261. [Abstract] [Full Text] [PDF]
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P. Fang, Z. Wang, and M. S. Sachs Evolutionarily Conserved Features of the Arginine Attenuator Peptide Provide the Necessary Requirements for Its Function in Translational Regulation J. Biol. Chem., August 25, 2000; 275(35): 26710 - 26719. [Abstract] [Full Text] [PDF]
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