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Liza J Conrad

Publications and source records attributed to Liza J Conrad.

3 recordsLinked to original sources

Ac-immobilized, a stable source of Activator transposase that mediates sporophytic and gametophytic excision of Dissociation elements in maize.

We have identified and characterized a novel Activator (Ac) element that is incapable of excision yet contributes to the canonical negative dosage effect of Ac. Cloning and sequence analysis of this immobilized Ac (Ac-im) revealed that it is identical to Ac with the exception of a 10-bp deletion of sequences at the left end of the element. In screens of approximately 6800 seeds, no germinal transpositions of Ac-im were detected. Importantly, Ac-im catalyzes germinal excisions of a Ds element resident at the r1 locus resulting in the recovery of independent transposed Ds insertions in approximately 4.5% of progeny kernels. Many of these transposition events occur during gametophytic development. Furthermore, we demonstrate that Ac-im transactivates multiple Ds insertions in somatic tissues including those in reporter alleles at bronze1, anthocyaninless1, and anthocyaninless2. We propose a model for the generation of Ac-im as an aberrant transposition event that failed to generate an 8-bp target site duplication and resulted in the deletion of Ac end sequences. We also discuss the utility of Ac-im in two-component Ac/Ds gene-tagging programs in maize.

Base Sequence↗

Distribution of Activator (Ac) throughout the maize genome for use in regional mutagenesis.

A collection of Activator (Ac)-containing, near-isogenic W22 inbred lines has been generated for use in regional mutagenesis experiments. Each line is homozygous for a single, precisely positioned Ac element and the Ds reporter, r1-sc:m3. Through classical and molecular genetic techniques, 158 transposed Ac elements (tr-Acs) were distributed throughout the maize genome and 41 were precisely placed on the linkage map utilizing multiple recombinant inbred populations. Several PCR techniques were utilized to amplify DNA fragments flanking tr-Ac insertions up to 8 kb in length. Sequencing and database searches of flanking DNA revealed that the majority of insertions are in hypomethylated, low- or single-copy sequences, indicating an insertion site preference for genic sequences in the genome. However, a number of Ac transposition events were to highly repetitive sequences in the genome. We present evidence that suggests Ac expression is regulated by genomic context resulting in subtle variations in Ac-mediated excision patterns. These tr-Ac lines can be utilized to isolate genes with unknown function, to conduct fine-scale genetic mapping experiments, and to generate novel allelic diversity in applied breeding programs.

Chromosome Mapping↗

Transposon tagging using Activator (Ac) in maize.

The transposable element Activator (Ac) has been used in several plant species as a tool for gene isolation and characterization. However, it has not been widely utilized in its native host maize, in part, because of a relatively low germinal transposition rate. The propensity of Ac to move to linked sites provides an opportunity to overcome this limitation when Ac elements are distributed at regular intervals throughout the genome. This chapter details the use of such a system in maize through simple genetic manipulations. A detailed protocol is also provided to clone DNA flanking Ac insertions.

Base Sequence↗