Site-selected transposon mutagenesis at the hcf106 locus in maize

Das, L., Martienssen, R. (March 1995) Site-selected transposon mutagenesis at the hcf106 locus in maize. Plant Cell, 7 (3). pp. 287-294. ISSN 1040-4651

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The High chlorophyll fluorescence106 (Hcl106) gene in maize is required for chloroplast membrane biogenesis, and the hcl106-mum1 allele is caused by the insertion of a Robertson's Mutator Mu1 element into the promoter of the gene, Seedlings homozygous for hcl106-mum1 are pale green and die 3 weeks after germination, but only in the presence of Muta for 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 hcl106 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 hcl106-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 hcl106-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 hcl106-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.

Item Type: Paper
Uncontrolled Keywords: dna modification caenorhabditis-elegans somatic instability mutator activity element system bz2-mu1 allele zea-mays gene drosophila expression
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification
organism description > plant > maize
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > transposons
CSHL Authors:
Communities: CSHL labs > Martienssen lab
Depositing User: Matt Covey
Date: March 1995
Date Deposited: 13 Dec 2013 16:38
Last Modified: 13 Dec 2013 16:38
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