No abstract

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 ϳ32-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 The Mutator family of transposable elements of Zea mays is considered to be one of the most efficient gene-tagging systems in eukaryotes. Mutator (Mu) transposable elements were first recognized by Robertson in a line of maize that exhibited a 50-to 100-fold increase in its spontaneous mutation frequency compared to standard maize stocks (45). Extensive genetic and molecular analysis has demonstrated that the increased mutation frequency is caused by the transposition of a family of Mu elements (reviewed in references 10, 13, and 16). Molecular analysis of Mu-induced mutations has identified at least eight classes of nonautonomous and autonomous Mu elements (10). These elements share highly conserved ϳ210-bp terminal inverted repeats (TIRs), and their insertion sites are flanked by a 9-bp direct repeat duplication of the host sequence. Elements are classified by the sequence similarity of their internal sequences. Mu1 is the most active Mu element as measured by frequency of insertion into tagged and cloned maize genes (13). Mu1 elements are also among the smallest of the Mu elements; they have ϳ210-bp TIRs encompassing an ϳ1,000-bp internal region (5). Mu1, like most of the elements in the Mu family, is a nonautonomous element that depends on the presence of MuDR (also called Mu9), the autonomous element, for its transposition (20,28,29,43).MuDR is a 4.9-kb element with characteristic Mu TIRs of ϳ210 bp, but it is unique in encoding proteins required for transposition. MuDR encodes two major transcripts, mudrA and mudrB, that are convergently transcribed from promoters in each of the TIRs. The mudrA and mudrB transcripts can be differentially spliced (27). It is not yet known if these splicing variants produce all of the predicted alternate protein products and, if so, what roles these protein products play in Mutator activities. This study focuses on the 823-amino-acid open reading frame, MURA, encoded by the major, fully spliced mudrA transcript (27). MURA is of particular interest because it shares a sequence motif with a family of bacterial insertion sequence transposases (22)...

The Mu transposons of maize are under stringent developmental control. Elements excise at high frequencies in terminally dividing somatic cells, but not in meristems. Mu elements in germinal cells amplify, without excision, and insert throughout the genome. All activities require MuDR, which encodes two genes, mudrA and mudrB, whose near-identical promoters are located in the transposon terminal inverted repeats (TIR). We have fused the 216 bp TIR of the mudrB gene to GUS and luciferase reporters. We demonstrate that TIRB programs reporter expression in diverse, meristematic somatic cells, paradoxically in those cells in which Mu excisions are repressed. In germinal cells, immature tassel and mature pollen, reporter expression increases up to 20-fold compared to leaf. By RNA blot hybridization, we demonstrate that endogenous mudrB and mudrA transcripts increase significantly in mature pollen; sequence comparisons demonstrate that the MuDR TIRs contain plant cell-cycle enhancer motifs and functionally defined pollen enhancers. Therefore, the MuDR TIR promoters are developmentally regulated in both somatic and germinal tissues. Because database sequence analysis suggests that the MuDR TIR enhancers should be functional in both monocots and dicots, we suggest that the native MuDR promoter be used in attempts to transfer the unique behavior of Mu transposition to heterologous hosts.

The mechanisms of intron recognition and processing have been well-studied in mammals and yeast, but in plants the biochemistry of splicing is not known and the rules for intron recognition are not clearly defined. To increase understanding of intron processing in plants, we have constructed new pairs of vectors, pSuccess and pFail, to assess the efficiency of splicing in maize cells. In the pFail series we use translation of pre-mRNA to monitor the amount of unspliced RNA. We inserted an ATG codon in the Bz2 (Bronze-2) intron in frame with luciferase: this construct will express luciferase activity only when splicing fails. In the pSuccess series the spliced message is monitored by inserting an ATG upstream of the Bz2 intron in frame with luciferase: this construct will express luciferase activity only when splicing succeeds. We show here, using both the wild-type Bz2 intron and the same intron with splice site mutations, that the efficiency of splicing can be estimated by the ratio between the luciferase activities of the vector pairs. We also show that mutations in the unique U-rich motif inside the intron can modulate splicing. In addition, a GC-rich insertion in the first exon increases the efficiency of splicing, suggesting that exons also play an important role in intron recognition and/or processing.

Summary The Mu transposons of maize are under stringent developmental control. Elements excise at high frequencies in terminally dividing somatic cells, but not in meristems. Mu elements in germinal cells amplify, without excision, and insert throughout the genome. All activities require MuDR, which encodes two genes, mudrA and mudrB, whose near‐identical promoters are located in the transposon terminal inverted repeats (TIR). We have fused the 216 bp TIR of the mudrB gene to GUS and luciferase reporters. We demonstrate that TIRB programs reporter expression in diverse, meristematic somatic cells, paradoxically in those cells in which Mu excisions are repressed. In germinal cells, immature tassel and mature pollen, reporter expression increases up to 20‐fold compared to leaf. By RNA blot hybridization, we demonstrate that endogenous mudrB and mudrA transcripts increase significantly in mature pollen; sequence comparisons demonstrate that the MuDR TIRs contain plant cell‐cycle enhancer motifs and functionally defined pollen enhancers. Therefore, the MuDR TIR promoters are developmentally regulated in both somatic and germinal tissues. Because database sequence analysis suggests that the MuDR TIR enhancers should be functional in both monocots and dicots, we suggest that the native MuDR promoter be used in attempts to transfer the unique behavior of Mu transposition to heterologous hosts.