The long term goal of this study is to understand how the Mutator transposable element activities are regulated within the life cycle of its natural host, maize. The Mutator system contains the autonomous MuDR element in addition to diverse non-autonomous Mutator transposons sharing only the ~200bp terminal inverted repeats (TIRs). Mobilization of the Mu elements by MuDR produces the highest forward mutation frequency of any eukaryotic transposon. Most striking is the alternative transposition mechanisms exhibited by the Mutator system: cut or cut and paste in differentiated somatic cells and replicative transposition in germ cells. Consistent with this complex lifestyle is an extraordinary complexity of potential and actual MuDR RNA and protein products. Convergent mudrA and mudrB transcripts initiate at promoters within the TIRs and terminate at repetitive intergenic sequences. MuRA has homology with bacterial transposases, MuRB is novel. A natural MuDRB deletion leads to fewer or no somatic excision. Constitutive expression of mudrA and B only in MuDR-containing lines has led Walbot to hypothesize that post-transcriptional events are responsible for the developmental late activation of Mutator and the alternative use of cut and paste and replicative mechanisms. Post-transcriptional mechanisms may include (i) modulation of the ratio of mudrA,B transcription by antisense RNA, (ii) translational control mediated by the long, AUG-containing leader of mudrA, (iii) developmental control of alternative splicing producing different MURA,B proteins and (iv) alternative polyadenylation sites producing different 3'UTRs that could influence RNA stability.
Four specific aims comprise this proposal. The first is to define which MuDR transcripts and proteins are present before and after Mutator activation in somatic and germinal cells. For this purpose, specific antisera has or will be raised to the six possible MURA and B proteins that result from alternative splicing. The possibility that alternative splicing is developmentally regulated will be tested. Transient expression assays are proposed to determine whether one of the two mudrA transcripts, with an upstream AUG, is regulated at the level of translation initiation. Finally, 14 lines of transgenic maize expressing (i) individual MuDR products including antisense or (ii) myc-tagged MuDR products or (iii) TIR reporter constructs have been or will be constructed and assayed for their ability to promote aspects of Mutator activity either alone or after crossing to appropriate lines.
The second aim i s to characterize the antisense products, see if they are developmentally regulated, and assess the effect of induction in transgenic plants.
The third aim i s study the mechanism of transposition in vivo using transgenic plants containing plasmids with marked Mu elements where both the donor site and new insertions can be recovered and analyzed. This assay system will be used to address such questions as (i) does efficient gap repair early in development mask excision and is excision accompanied by new insertions, (ii) do all Mu elements participate in replicative transposition, (iii) which MuDr products prevent TIR methylation, and (iv) what combination of MuDR products is necessary to promoter somatic vs. germinal transposition? The fourth and last aim focuses on an in vitro analysis of MURA/B binding to DNA templates, their nuclear localization and their interactions with each other and with methylated substrates.
| Eisen, J A; Benito, M I; Walbot, V (1994) Sequence similarity of putative transposases links the maize Mutator autonomous element and a group of bacterial insertion sequences. Nucleic Acids Res 22:2634-6 |
