Ac-Immobilized, a Stable Source of Activator Transposase That Mediates Sporophytic and Gametophytic Excision of Dissociation Elements in Maize

Abstract

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 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 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.

Generating Novel Allelic Variation ThroughActivatorInsertional Mutagenesis in Maize

“…As 36 seedlings were found to carry both Ac insertions, we calculate a minimal frequency of nonconcordance at 11%. For comparison, $40% of all Ds excisions from the r1 locus mediated by Ac-im occur during gametophytic development (Conrad and Brutnell 2005) whereas at least 34% of Ac transpositions from the bz-m2(Ac) likely occur at a cell division during megagametogenesis (Dooner and Belachew 1989). Thus, our calculated frequency of nonconcordance is likely to be an underestimate of the true frequency of gametophytic transposition.…”

State IIDissociationElement Formation FollowingActivatorExcision in Maize

“…Increasing copy numbers of Ac in the genome result in fewer and smaller sectors known as the negative dosage effect (McClintock 1951). The Ac-immobilized (Ac-im) stock contains an Ac derivative inserted at 7.02, which is incapable of transposition but still encodes a functional transposase protein (Conrad and Brutnell 2005).…”

“…PCR primers designed for Ac insertion verification described in Kolkman et al (2005) were utilized to amplify a region from the DNA flanking the element into the end of putative Ds derivatives (supplemental Table 1 at http:/ /www.genetics.org/supplemental/). Verification PCR was performed using 6 ng of total genomic DNA in a 50 ml total reaction volume according to reaction conditions described in Conrad and Brutnell (2005). PCR products were fractionated on a 0.8% agarose gel containing ethidium bromide and visualized on a UV light box.…”

“…To demonstrate this method, a plant carrying the Ds insertion bti00252TDs(D2) was crossed by an individual homozygous for Ac-im (materials and methods). Ac-im is a stabilized source of transposase, capable of mediating somatic and germinal Ds excisions (Conrad and Brutnell 2005). PCR primers were designed to sequences flanking the Ds insertion (GenBank accession no.…”

“…When mutable phenotypes are desirable, for instance, in clonal analysis (Emerson 1917;Dawe and Freeling 1990) or to generate an allelic series (Wessler et al 1986;Moreno et al 1992;Weil et al 1992;Singh et al 2003), the elements can be remobilized by introducing an Ac element into the genome through crossing. Unfortunately, relatively few Ds insertions have been genetically or physically mapped in the maize genome, limiting the potential of Ds in directed or regional tagging programs (Conrad and Brutnell 2005).…”