Academic journal article Genetics

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

Academic journal article Genetics

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

Article excerpt

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.

IN the absence of efficient gene replacement technologies, plant geneticists have relied heavily on insertional mutagenesis to elucidate gene function. In maize, several groups have worked to distribute the maize transposable element Activator (Ac) throughout the genome for use in forward genetic screens (UELLAPORTA and MORENO 1994; DOONER et al. 1994; AUGER and SHERIDAN 1999; KOLKMAN et al. 2005). As Ac tends to transpose to closely linked sites, elements inserted near a gene of interest can be used as donors in regional mutagenesis programs (DELLAPORTA et al. 1988; DELONG et al. 1993; SHEN et al. 2000; SINGH et al. 2003). The propensity for short-range transposition (GREENBLATT 1984; DOONER and BELACHEW 1989) makes it likely that multiple insertion alleles will be recovered (SiNGH et al. 2003) and a high frequency of germinal reversion associated with Ac-induced alleles can be exploited to generate stable excision alleles (MA and DOONER 2004). Importantly, many Ac elements have been distributed in a uniform genetic inbred, greatly facilitating the analysis of recovered mutants by permitting nearisogenic comparisons of mutant and wild-type individuals (CowpERTHWAiTE et al. 2002; KOLKMAN el al. 2005).

Despite these many attractive features of Ac, there are several practical limitations of Ac for large-scale tagging programs. For one, relatively few mapped Ac elements are positioned in reporter genes (DELLAPORTA and MORENO 1994; AUGER and SHERIDAN 1999). Instead, most have been placed relative to a translocation breakpoint (UOONER et al. 1994) or relative to molecular markers on recombinant inbred populations (KOLKMAN et al. 2005). As a result, the integrity of these lines can be confirmed only through time-consuming mapping experiments or by a molecular assay. In addition, propagating lines containing active Ac insertions over several generations is likely to lead to an increased mutational load due to transposition of the autonomous Ac element or nonautonomous Ds insertions present in the genome. The frequency of germinal Ac transposition events is also relatively low, averaging from 2 to 4% of kernel progeny (UOONER and BELACHEW 1989; BRUTNELL and DELLAPORTA 1994). Thus, given the low number of precisely positioned Ac insertions available, a large number of crosses must be performed to ensure a high probability that an insertion will be recovered in a gene of interest (BRUTNELL and CONRAD 2003).

A two-component Ac/Ds system in maize would circumvent many of these limitations. If Ds insertion lines lacking active Ac elements are crossed by an Ac transposase source that is incapable of excision, Ds transpositions could be generated and the resulting Ds insertion alleles maintained as either unstable ( + Ac) or stable (- Ac) alleles. A primary limitation to date has been the lack of a genetically well-characterized source of immobilized Ac transposase that is capable of mediating a high frequency of Ds excision in maize. …

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