Mu9, a new 5 kb member of the Mutator family of transposable elements

--Jane Hershberger, Christine Anne Warren and Virginia Walbot

The Mutator family of transposable elements causes high rates of forward mutation. Eight elements, named Mu1 through Mu8, have been previously characterized. They range from 1.0 - 2.2 kb in size, and they have unique central regions flanked by homologous 215 bp terminal inverted repeats (TIRs). In a recent screening for new Mutator-induced insertions in genes of the anthocyanin pathway, this laboratory isolated bz2-mu4, an unstable allele of the Bronze-2 gene. Like many other Mutator insertions, bz2-mu4 gives rise to small, frequent sectors of somatic excision and infrequent germinal revertants. Southern analysis of this mutant using probes from Bz2 revealed the presence of a large (approximately 5 kb) insertion in the second exon of Bz2. To determine whether this large insertion was related to other large transposable elements, we digested genomic bz2-mu4 DNA with a panel of diagnostic restriction endonucleases and blotted it. From the derived restriction map it was clear that the insertion was neither Ac, Spm/En, nor any simple deletion derivative of these elements. Hybridizing the same blots with a probe from the 215 bp TIR of the Mu1 element did not, however, conclusively prove that this insertion belonged to the Mutator family of transposable elements. Therefore, we cloned the insertion to analyze it in more detail.

The insertion, flanked by about 5 kb of Bz2 sequences, was cloned on two EcoRI fragments from a lambda zap II (Stratagene) genomic library. The restriction map of the isolated clones matches the one derived from our analysis of the genomic Southern blots. We have used several fragments derived from these clones to examine the copy number of this insertion and its derivatives in the maize genome. On Southern blots of maize genomic DNA, both active Mutator lines and non-Mutator lines contain multiple hybridizing fragments. A 500 bp probe from the central region of the cloned insertion hybridizes to 5-10 bands per haploid genome; a 450 bp fragment adjacent to the 3' TIR hybridizes to 10-15 bands per haploid genome. Thus, it appears that this sequence or deletion derivatives thereof exist in most or all lines.

Sequence analysis of the cloned DNA indicates that this 5 kb insertion from bz2-mu4 is a new member of the Mutator family of transposable elements; we have named it Mu9. The insertion is flanked by 9 bp direct repeats of the sequence TCCTGGAGG. Mu9 has the characteristic 215 bp TIR of the Mutator family, and these are 80-90% similar to the TIRs of other known Mutator elements. The Mu1 TIR that was used as a probe on the genomic Southern blots mentioned above is only about 82% similar to the Mu9 TIR, thus, hybridization between the two TIRs would be weak under the high stringency wash conditions we used.

As the sequence analysis of Mu9 is still in progress we cannot yet draw firm conclusions about the protein(s) encoded by this element. However, multiple open reading frames of 100 amino acids or more are present throughout the sequence. We will use both RNAse protection and cDNA cloning to determine the exon/intron structure of Mu9. The previously identified Mutator elements do not have long open reading frames; because Mu9 is more than twice as large as any of these, it has the potential to encode proteins that may be involved in the transposition process. Computer analysis should enable us to identify DNA-binding regions and other functional domains if any are present in the Mu9-encoded protein. Because Mu9 has transposed into Bz2, we will be able to analyze this element and any proteins it encodes at both the molecular and the genetic level.

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