The mop1 (mediator of paramutation1) mutant progressively reactivates one of the two genes encoded by the MuDR transposon in maize

Transposons make up a sizable portion of most genomes, and most organisms have evolved mechanisms to silence them. In maize, silencing of the Mutator family of transposons is associated with methylation of the terminal inverted repeats (TIRs) surrounding the autonomous element and loss of mudrA expression (the transposase) as well as mudrB (a gene involved in insertional activity). We have previously reported that a mutation that suppresses paramutation in maize, mop1, also hypomethylates Mu1 elements and restores somatic activity to silenced MuDR elements. Here, we describe the progressive reactivation of silenced mudrA after several generations in a mop1 background. In mop1 mutants, the TIRA becomes hypomethylated immediately, but mudrA expression and significant somatic reactivation is not observed until silenced MuDR has been exposed to mop1 for several generations. In subsequent generations, individuals that are heterozygous or wild type for the Mop1 allele continue to exhibit hypomethylation at Mu1 and mudrA TIRs as well as somatic activity and high levels of mudrA expression. Thus, mudrA silencing can be progressively and heritably reversed. Conversely, mudrB expression is never restored, its TIR remains methylated, and new insertions of Mu elements are not observed. These data suggest that mudrA and mudrB silencing may be maintained via distinct mechanisms.     

Mutator transposase is widespread in the grasses

Although the Mutator (Mu) system is well characterized in maize (Zea mays), very little is known about this highly mutagenic system of transposons in other grasses. Mutator is regulated by the MuDR class of elements, which encodes two genes, one of which, mudrA, has similarity to a number of bacterial transposases. Experiments in our laboratory, as well as database searches, demonstrate that mudrA sequences are ubiquitous and diverse in the grasses. In several species it is clear that multiple paralogous elements can be present in a single genome. In some species such as wheat (Triticum aestivum) and rice (Oryza sativa), mudrA-similar sequences are represented in cDNA databases, suggesting the presence of active Mu transposon systems in these species. Further, in rice and in sorghum, mudrA-like genes are flanked by long terminal inverted repeats, as well as the short host sequence direct repeats diagnostic of insertion. Thus, there is ample evidence that systems related to Mu in maize are at least potentially active in a wide variety of grasses. However, the mudrB gene, though important for Mu activity in maize, is not necessarily a component of Mu elements in other grasses.     

Characterization of the maize Mutator transposable element MURA transposase as a DNA-binding protein.

The autonomous MuDR element of the Mutator (Mu) transposable element family of maize encodes at least two proteins, MURA and MURB. Based on amino acid sequence similarity, previous studies have reported that MURA is likely to be a transposase. The functional characterization of MURA has been hindered by the instability of its cDNA, mudrA, in Escherichia coli. In this study, we report the first successful stabilization and expression of MURA in Saccharomyces cerevisiae. Gel mobility shift assays demonstrate that MURA is a DNA-binding protein that specifically binds to sequences within the highly conserved Mu element terminal inverted repeats (TIRs). DNase I and 1,10-phenanthroline-copper footprinting of MURA-Mu1 TIR complexes indicate that MURA binds to a conserved approximately 32-bp region in the TIR of Mu1. In addition, MURA can bind to the same region in the TIRs of all tested actively transposing Mu elements but binds poorly to the diverged Mu TIRs of inactive elements. Previous studies have reported a correlation between Mu transposon inactivation and methylation of the Mu element TIRs. Gel mobility shift assays demonstrate that MURA can interact differentially with unmethylated, hemimethylated, and homomethylated TIR substrates. The significance of MURA's interaction with the TIRs of Mu elements is discussed in the context of what is known about the regulation and mechanisms of Mutator activities in maize.     

The <Emphasis Type="Italic">piggyBac</Emphasis> Transposon as a Tool in Genetic Engineering

Transposons are mobile genetic elements that are part of the genomic DNA of numerous organisms and belong to two classes. Unlike class I transposons, class II DNA transposons do not use the stage of RNA synthesis in their transition; they perform it by the cut-and-paste mechanism or with a replicative transposition. The integration of a DNA transposon in a new site results in the duplication of a target sequence on either side of a transposon, and its excision is, as a rule, associated with insertions and deletions. The piggyBac transposon isolated from the Trichoplusia ni moth differs from other mobile elements of its class. Due to its unique ability to leave no traces after excision from an insertion site and to perform successful transposition and transference of large DNA fragments, piggyBac is a convenient tool for the development of gene engineering approaches. The TTAA sequence serves as a target site for transposon integration: insertion in the AT-rich DNA regions is more frequent. The ability of piggyBac to be transferred to a new area independently of the cell apparatus and to restore a DNA site without error after excision lies in the mechanism of its transposition, which is discussed in detail in the present review. Along with other transposons and viruses, the piggyBac transposon is widely used in the transgenesis of various organisms; it also finds application in insertion mutagenesis and gene therapy.     

MuDR transposase increases the frequency of meiotic crossovers in the vicinity of a Mu insertion in the maize a1 gene

Although DNA breaks stimulate mitotic recombination in plants, their effects on meiotic recombination are not known. Recombination across a maize a1 allele containing a nonautonomous Mu transposon was studied in the presence and absence of the MuDR-encoded transposase. Recombinant A1' alleles isolated from a1-mum2/a1::rdt heterozygotes arose via either crossovers (32 CO events) or noncrossovers (8 NCO events). In the presence of MuDR, the rate of COs increased fourfold. This increase is most likely a consequence of the repair of MuDR-induced DNA breaks at the Mu1 insertion in a1-mum2. Hence, this study provides the first in vivo evidence that DNA breaks stimulate meiotic crossovers in plants. The distribution of recombination breakpoints is not affected by the presence of MuDR in that 19 of 24 breakpoints isolated from plants that carried MuDR mapped to a previously defined 377-bp recombination hotspot. This result is consistent with the hypothesis that the DNA breaks that initiate recombination at a1 cluster at its 5' end. Conversion tracts associated with eight NCO events ranged in size from <700 bp to >1600 bp. This study also establishes that MuDR functions during meiosis and that ratios of CO/NCO vary among genes and can be influenced by genetic background.     

Somatic excision of the Mu1 transposable element of maize.

The Mu transposons of the Robertsons's Mutator transposable element system in maize are unusual in many respects, when compared to the other known plant transposon systems. The excision of these elements occurs late in somatic tissues and very rarely in the germ line. Unlike the other plant transposons, there is no experimental evidence directly linking Mu element excision and integration. We have analyzed the excision products generated by a Mu1 transposon inserted into the bronze 1 locus of maize. We find that the excision products or 'footprints' left by the Mu1 element resemble those of the other plant transposable elements, rather than those of the animal transposable element systems. We also find some novel types of footprints resembling recombinational events. We suggest that the Mu1 element can promote intrachromosomal crossovers and conversions near its site of insertion, and that this may be another mechanism by which transposons can accelerate the evolution of genomes.