University of Florida

Ds revertant alleles from sh2-m1 show a high frequency of precise excision
--Michael J. Giroux, Ronald J. Okagaki and L. Curtis Hannah

We are currently analyzing wildtype revertants of a mutable Ds containing allele, sh2-m1. Revertants from this allele have been shown to differ in their allosteric properties for the enzyme ADPglucose pyrophosphorylase (AGP).

AGP in maize is encoded by the shrunken2 (sh2) and brittle2 (bt2) genes. AGP is believed to be the rate limiting step in starch biosynthesis and is an allosteric enzyme activated by 3-PGA and inhibited by PO4.

The site of Ds insertion in the sh2-m1 allele is in a region important for the regulation of AGP in plants. Previous data have shown that revertants from the sh2-m1 allele differ in their allosteric properties. The Ds element in sh2-m1 is located 9 amino acids from a lysine residue important in the regulation of the spinach AGP enzyme (Figure, part A). This region is also highly conserved among different plant species. The Ds is only 20 amino acids from the 3' end of the SH2 protein in the gene's last exon.

The DNA sequence of the revertant alleles at the site of Ds insertion was characterized in 27 revertant alleles by direct DNA sequencing of PCR products. The PCR products were amplified with primers flanking a 400 base pair region surrounding the site of Ds insertion. Both strands of the PCR products were sequenced and the revertant sequence data is summarized in the Figure, part B.

Approximately 50% of the revertants (12/27) contain no Ds footprint. Of the remainder, roughly 50% (11/27) contain a footprint of TAC (tyrosine). This likely represents a duplication of host sequences during repair synthesis following excision of the Ds element.

Several exceptional sequences were also noted. Two of the 27 revertants sequenced contain a footprint of CCT. One revertant has the added sequence CGTACT and one, TTACTA. These sequences are not explained by current models of transposable element transposition in plants. These models postulate footprints consisting of a portion of the target site duplication or a wildtype sequence. While most of our sequenced revertants are explained as portions of the duplicated host sequence or perfect excisions, several are not. The high frequency of perfect excisions also contradicts evidence that the perfect excision of transposable elements is rare. However, this may reflect the importance of this region of the Sh2 gene to AGP activity. This is supported by the fact that each of the germinal revertants analyzed restores the wildtype open reading frame. 

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