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THE PLANT CELL, Vol 7, Issue 3 287-294, Copyright © 1995 by American Society of Plant Biologists
Site-Selected Transposon Mutagenesis at the hcf106 Locus in Maize
L. Das and R. Martienssen
Cold Spring Harbor Laboratory, Box 100, Cold Spring Harbor, New York 11724
The High chlorophyll fluorescence106 (Hcf106) gene in maize is required for
chloroplast membrane biogenesis, and the hcf106-mum1 allele is caused by
the insertion of a Robertson's Mutator Mu1 element into the promoter of the
gene. Seedlings homozygous for hcf106-mum1 are pale green and die 3 weeks
after germination, but only in the presence of Mutator activity conferred
by active, autonomous Mu regulatory transposons elsewhere in the genome.
When Mutator activity is lost, the mutant phenotype is suppressed, and
homozygous plants have an almost wild-type phenotype. To isolate derivative
alleles at the hcf106 locus that no longer require Mutator activity for
phenotypic expression, we have developed a method for site-selected
transposon mutagenesis in maize. This procedure, first described for
Caenorhabditis elegans and Drosophila, involves using polymerase chain
reaction (PCR) to screen pools of individuals for insertions and deletions
in genes of known sequence. Pools of seedlings segregating for the
progenitor allele hcf106-mum1 were screened by PCR for insertions and
deletions associated with Robertson's Mutator. In a 360-bp target region,
two new insertions and one deletion were identified in only 700 Mu-active
gametes screened. One of the insertions was in the progenitor hcf106-mum1
allele and the other was in the wild-type allele, but all three new alleles
were found to have breakpoints at the same nucleotide in the first intron.
Unlike the hcf106-mum1 progenitor allele, the deletion and one of the
insertions conferred pale green seedling lethal phenotypes in the absence
of Mutator activity. However, the second insertion had a weak, viable
phenotype under these conditions. Although the sample size was small, our
results suggest that this procedure can be used to rapidly identify
transposon insertions into known genes in a single generation. Null
derivative alleles can then be isolated in a second generation using the
insertion as a starting point. This two-step procedure represents a
powerful and simple way to "knock out" maize genes identified by sequence
alone by using only a few thousand progeny from a simple cross.
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