Mx-rMx, a family of interacting transposons in the growing hAT superfamily of maize

Abstract

More than half a century after the discovery of transposable elements, the number of genetically defined autonomous elements that have been isolated and characterized molecularly in any one species remains surprisingly small. Because of its rich genetic history, maize (Zea mays) is, by far, the plant with the largest number of such elements. Yet, even in maize, a maximum of only two autonomous elements have been characterized in any transposon superfamily. This article describes the isolation and molecular and genetic characterization of Mx (for mobile element induced by x-rays), a third autonomous member of the hAT transposon superfamily in maize. Mx is 3731 bp long, ends in 13-bp terminal inverted repeats (TIRs), and causes an 8-bp duplication of the target site. Mx and rMx (for responder to Mx), its 571-bp nonautonomous partner, define a classical family of interacting transposable elements. Surprisingly, the TIRs of Mx and rMx are only 73% identical, and the subterminal sequences are even less so, suggesting that Mx and rMx may represent diverging transposable elements still capable of mobilization by the same transposase. Sequences that are closer to the ends of either Mx or rMx are present in the maize genome. Mx is predicted to encode a 674-amino acid protein that is homologous to the Ac transposase. Although Mx and Ac are closely related, they do not interact. Other data suggest that maize may possess at least five families of hAT transposons that do not interact with each other. The possible origin of noninteracting transposon families within the same superfamily is discussed.

Figure 1.

bz-x3m Phenotypes. (A) Ear of a W22 bz-x3m homozygote, showing the finely spotted kernel phenotype arising from late somatic reversions in the aleurone layer. A few kernels show larger sectors produced from earlier reversion events. (B) Close-up of (A), showing the small size of the majority of revertant sectors in the aleurone. (C) Spotted and red anthers in a W22 bz-x3m homozygote. Most reversions occur late in development and result in very finely spotted anthers. However, reversions can also occur earlier, as indicated by the two florets with exclusively red anthers.

Figure 2.

DNA Sequence Comparison between rMx and Tz86. The entire 571-bp rMx element from bz-x3m was compared with the Tz86 ends. The Tz86 sequence is a 439-nucleotide composite, consisting of 207 nucleotides from the 5′ end (GenBank accession M10174) and 232 nucleotides from the 3′ end (GenBank accession M10175). Sequences were aligned using the program MultiAlin (http://prodes.toulouse.inra.fr/multalin/multalin.html). The graphical output highlights identical nucleotides as white letters in black boxes and purine transitions as white letters in gray boxes (Box-Shader 3.21, http://www.ch.embnet.org/software/BOX_form.html).

Figure 3.

Genetic Scheme to Separate Mx and rMx in the W22 bz-x3m Stock. See text for details.

Figure 4.

Primer Walking to Isolate Mx. (A) Sequential PCR steps to amplify progressively larger members of the Mx family. p1 to p6 stand for specific primers used in the amplification reactions. Boxes with same shading indicate identical or highly similar sequences. (B) Ethidium bromide (EtBr)–stained agarose gel of PCR amplification products with primers p1 and p2. Lane 1, 1-kb ladder of molecular size markers; lane 2, PCR products from Mx bz-x3m, showing the 0.8- and 0.5-kb bands, corresponding to dMx1 and rMx, respectively. (C) EtBr-stained agarose gel of PCR amplification products with primers p3 and p4. Lane 1, 1-kb ladder of molecular size markers; lane 2, PCR products from Mx bz-x3m, showing the 2.2-kb band corresponding to dMx2. (D) EtBr-stained agarose gel of PCR amplification products with primers p5 and p6. Lane 1, 1-kb ladder of molecular size markers; lane 2, PCR products from Mx bz-x3m, showing the 3.0-kb band corresponding to the putative Mx element; lane 3, PCR products from W22 bz-R.

Figure 5.

DNA Gel Blot Analysis of Recombinants from the W22 Mx bz-x3m Stock. Reciprocal recombinants from Mx Sh bz-x3m/+ sh Bz heterozygotes were obtained by the scheme diagrammed in Figure 3. Genomic DNA was digested with EcoRI, blotted onto Nylon membranes, and hybridized sequentially to radiolabeled probes from dMx2 (A) (Figure 4A) and tmx (B), the Mx flanking region. P1, Mx Sh bz-x3m wx parent; P2, sh Bz Wx parent; P3, sh bz-R wx testcross parent; lanes 1 to 7, Mx Sh bz-R wx/+ sh bz-R wx recombinants; lanes 8 to 13, + sh bz-x3m wx/+ sh bz-R wx recombinants. The arrowhead identifies the position of Mx.

Figure 6.

DNA Gel Blot Analysis of Exceptions from Mx bz-x3m That Have Lost Mx. Genomic DNA was digested with EcoRI, blotted onto nylon membranes, and hybridized to a radiolabeled probe from dMx2. Lane 1, the Mx tester line + sh bz-x3m wx; lane 2, Mx Sh bz-x3m; lanes 3 to 5, three + Sh bz-x3m derivatives from Mx Sh bz-x3m. The arrowhead identifies the position of Mx.

Figure 7.

Mx Structure and Sequence Comparison. (A) Structure of Mx, showing the location of the 13-bp TIRs, the subterminal hexanucleotide repeats, and the exons and introns of its predicted protein, Mx TPase. This protein is homologous to the transposases encoded by Ac and other members of the hAT transposon superfamily, which share three conserved domains, indicated as hAT1, hAT2, and hAT3. (B) Amino acid sequence alignment of the putative transposases encoded by Mx, Ac, and Tam3. The latter two are the main representatives of autonomous hAT elements from monocots and dicots, respectively. The sequences were aligned with MultiAlin, and the aligned sequences were decorated with BoxShade 3.21. Identity and similarity of amino acid residues are indicated by black and gray shading, respectively.

Figure 8.

DNA Gel Blot Analysis of Mx Transposition. Genomic DNA was digested with NcoI, blotted onto Nylon membranes, and hybridized to an internal Mx probe extending from position 761 to 1710 in the nucleotide sequence. Lane 1, bz-x3m stock (Mx Sh bz-x3m); lane 2, Mx tester line (+ sh bz-x3m); lanes 3 to 6, derivatives of the bz-x3m stock with transposed Mx elements. Arrow, original Mx element; arrowheads, transposed Mx elements.

Figure 9.

DNA Gel Blot Analysis of Mx Abundance in Maize Inbreds and Wild Relatives. Genomic DNA was digested with NcoI, blotted onto Nylon membranes, and hybridized to the same internal Mx probe as in Figure 8. Lanes 1 and 2, Z. mays ssp mexicana, collected in Estado, Mexico; lanes 3 and 4, Z. mays ssp parviglumis, collected in Michoacán; lane 5, Z. mays ssp mexicana, collected in Michocán; lane 6, A188; lane 7, A636; lane 8, B73; lane 9, BSSS53; lane 10, 4Co63; lane 11, H99; lane 12, M14; lane 13, Mo17; lane 14, W22; lane 15, W23.

Figure 10.

Phylogenetic Tree of hAT Transposases. Sequences were aligned using ClustalX, and the tree was constructed using neighbor joining in MEGA version 2.1. Each sequence is identified by either its transposon name or GenBank accession number and the name of its source species. Plant autonomous elements are indicated in bold. Numbers above the branches indicate the percentage of 1000 bootstrap replications in which that branch was present. The well-supported clades are indicated by brackets and given the name of the first described element(s) in that clade.