abstract

[Matthew Sachs]

Genetics, in press

A UV-induced mutation in Neurospora that affects translational regulation
in response to arginine

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
isolated 46 mutants which 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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Matthew Sachs
Department of Chemistry, Biochemistry and Molecular Biology
Oregon Graduate Institute of Science and Technology
20000 NW Walker Road
P.O. Box 91000
Portland, OR  97291-1000
503 690-1487 Phone
503 690-1464 Fax
msachs at admin.ogi.edu