THE PLANT CELL, Vol 2, Issue 12 1225-1238, Copyright © 1990 by American Society of Plant Biologists
Insertion of Mu1 Elements in the First Intron of the Adh1-S Gene of Maize Results in Novel RNA Processing Events
K. R. Luehrsen and V. Walbot
Department of Biological Sciences, Stanford University, Stanford, California 94305
Maize transposable elements, when inserted in or near genes, alter
expression by several transcriptional and post-transcriptional mechanisms.
Three independent, unstable insertions of the transposable element Mutator
(Mu) into the first intron of the Alcohol dehydrogenase-1 (Adh1) gene have
been shown to decrease expression [Strommer et al. (1982). Nature
300,542-544]. We have developed an approach to elucidate the underlying
molecular mechanisms responsible for the mutant phenotypes. Mu1 elements
were inserted into Adh1-S intron 1 in vitro to create plasmid facsimiles of
the mutant alleles. The Mu1 element was also inserted at novel positions
within intron 1 to create new mutations. The Mu1/intron constructions were
placed between the Adh1-S promoter/exon 1 segment and a reporter gene
(firefly luciferase or [beta]-glucuronidase), and these chimeric gene
constructs were tested in transient assays in maize protoplasts. When
compared with the appropriate control, the Mu1 insertions decreased
reporter gene expression to levels approximating the alcohol dehydrogenase
enzyme activities observed for the Adh1-S mutants in vivo. The Mu1
insertions also showed a polarity effect with luciferase expression
increasing as the insertions were placed nearer the 3[prime] splice
junction. In addition, Mu1 insertions within a different intron, actin
intron 3, also significantly reduced luciferase expression, indicating that
Mu1 insertions within introns are likely to diminish expression in many
genes. The presence of the Mu1 sequences was correlated with decreased
levels of steady-state luciferase transcript. Deletion analysis of the Mu1
element and RNase mapping indicate that the transposable element contains
RNA processing signals in its central region that are largely responsible
for the decrease in expression.
