Dotted is a two-element transposon system consisting of the dotted element Dt, plus a second receptor element for Dt, rDt (M. Rhoades, J. Genetics 33:347-354, 1936). Dt not only controls its own transposition, but also controls the expression of several alleles of the anthocyanin locus, A1. The presence of the rDt element in these A1 alleles is inferred from their mutability in response to Dt (B. McClintock, Cold Spring Harbor Symp. Quant. Biol. 21:197-216, 1956). To address the mechanism of Dt-controlled A1 mutability, we cloned, sequenced, and determined the insertional position of rDt elements found in two mutable A1 alleles.
The alleles studied were a1, the standard recessive allele which probably originated from A-r (present in N. American races), and a-m-1:Cache, which originated from A:Cache (M.G. Neuffer, Genetics 46:625-640,1961). Both alleles are recessive in the absence of Dt, presumably due to insertional inactivation of A1 by the rDt element. These two alleles also differ in their response to Dt, a-m-I:Cache being more mutable.
To clone a1 and a-m-1:Cache, we constructed genomic libraries from plants carrying these alleles, and then screened these with an A1 probe (C. O'Reilly et al., EMBO J. 4:877-882, 1985). We characterized positive clones by restriction enzyme mapping and by nucleotide sequencing. These data, plus our previous data on the A1 gene itself (Zs. Schwarz-Sommer et al., EMBO J., in press), have allowed us to characterize the rDt element at the molecular level.
Comparisons of the restriction enzyme maps of A1, a1, and a-m-1:Cache identify similar 0.7 kb insertions in the latter two alleles. The sequences of the two insertions are very similar (>80% homology), but they are inserted at A1 in opposite orientations. The ends of the insertion contain 11 bp inverted-repeats, and the insertion is flanked by a 9 bp direct-repeat of A1 sequence in each case. The insertions in a-m-1:Cache and a1 interrupt the A1 coding sequence in exons 3 and 4 respectively, and each generates a translational stop signal.
Since we found insertions of similar nucleotide sequence in two Dt-responding A1 alleles of independent origin, we believe the insertions are the rDt controlling element. The rDt element is 0.7 kb long and has molecular characteristics typical of other maize controlling elements; Ac, Ds, and Spm (terminal inverted repeats flanked by a target site duplication). As expected, the recessive null phenotype of both alleles can be explained by the rDt insertion within the coding sequences of a1 and a-m1:Cache. In this respect rDt is similar to Ds and other transposons, which can cause insertional inactivation of the gene.
J.J. Sorrentino, C.O'Reilly1, Zs. Schwarz-Sommer2, H. Saedler2 and N.S. Shepherd
1Sunderland Polytechnic, Sunderland, UK.
2Max-Planck-Institut, Cologne, FRG.
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