University of Berkeley
Mu suppressible Liguleless3 alleles produce altered transcripts
--Lisa Girard, L, Freeling, M

Lg3-0 is a semi-dominant, neomorphic mutation that transforms regions of leaf blade, auricle and ligule into sheath. The Liguleless3 gene is a member of the knox class 1 family of homeobox genes and the dominant alleles which define it are due to ectopic expression of the gene in the leaf (Muehlbauer et al., Plant Physiology in press).

Screens for Mutator induced revertants of Lg3-0 resulted in the isolation of numerous partial and complete revertants in which the Lg3-0 phenotype is either reduced in severity or eliminated entirely. A subset of these revertant alleles were determined to be Mu suppressible. Mu suppression is when the phenotype caused by the Mu insertion is dependent on Mu activity such that in the absence of Mu activity, the phenotype reverts to that of the progenitor. In this context, when Mu is active the Lg3 suppressible alleles appear wild type and when Mu is inactive the plants appear mutant.

The Lg3-0r331, Lg3-0r422, and Lg3-0r1021 alleles are Mu suppressible revertants of Lg3-0. Each is caused by the insertion of a Mu element into the same site in the 5'UTR. We used Northern analysis to make an initial assessment of how these alleles might deal with a large, cumbersome insertion into their transcribed region. We found that the transcripts produced by these alleles are significantly shorter than those of wild type as well as its progenitor, Lg3-0.

In order to characterize these aberrant transcripts further, we used RACE (Rapid Amplification of cDNA Ends ) to clone the cDNA corresponding to the Lg3-0r422 transcript. We found that the transcripts produced by this allele are being initiated approximately 187 base pairs downstream relative to wild type (Fig.1).

The Lg3-0r331, Lg3-0r422, and Lg3-0r1021 alleles produce a transcript that is much smaller than that of wild type or its progenitor. Since all three of these alleles are Mu suppressible, we initially thought that they might be behaving in a manner similar to the suppressible hcf106, in which a Mu element inserted into the 5'UTR functions as an outward reading promoter. Instead, characterization of the Lg3-0r422 cDNA revealed that in this allele, transcription is initiated much further downstream than the site of Mu insertion, as if the element were able to redirect the start of transcription. One possibility is that the insertion causes the adoption of a secondary structure in the region which is prohibitive to transcription initiation at the correct site.

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