Matthew Sachs msachs at ADMIN.OGI.EDU
Mon Oct 9 12:14:36 EST 1995

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.

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

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