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THE PLANT CELL, Vol 2, Issue 3 255-261, Copyright © 1990 by American Society of Plant Biologists
Expression Analysis of a Pseudogene in Transgenic Tobacco: A Frameshift Mutation Prevents mRNA Accumulation
T. A. Voelker, J. Moreno and M. J. Chrispeels
Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0116
Seeds of the Pinto cultivar of the common bean, Phaseolus vulgaris, are
deficient in phytohemagglutinin (PHA), a lectin normally composed of two
different polypeptides (PHA-E and PHA-L). In Pinto seeds, there is no PHA-E
and only small amounts of PHA-L. The gene coding for the Pinto PHA-E,
Pdlec1, is a pseudogene as a result of a single base pair deletion in codon
11, causing a frameshift and premature termination of translation. This
mutation explains the absence of the PHA-E polypeptide but not the
several-hundredfold reduction of the cytoplasmic Pdlec1 mRNA in developing
seeds when compared with a normal PHA-E gene. To find the cause for this
reduction in mRNA levels, we swapped gene fragments of Pdlec1 with the
homologous parts of a normal PHA gene from the cultivar Greensleeves and
introduced these fusions into tobacco. Analysis of the transgenic seeds
showed that the Pdlec1 promoter is fully functional. We also repaired the
Pdlec1 coding frame in vitro and inserted the repaired and unrepaired
versions into a PHA gene expression cassette. In transgenic tobacco, both
constructs showed Pdlec1 transcript accumulation in the second half of seed
maturation. The single-base frame repair boosted the peak transcript levels
by a factor of 40 and resulted in the synthesis of PHA-E at normal levels.
We propose that the premature translational stop caused by the frameshift
leads to a faster breakdown of the Pdlec1 mRNA, thereby preventing this
transcript from accumulating to high levels.
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