Normally in our minimal line, which carries a single MuDR element at a known position (MuDR(p1)) and a single Mu1 element, methylation only occurs in progeny that lack a functional MuDR element. In families segregating Mu Killer, however, half the progeny that carry MuDR(p1) have methylated Mu1 TIRs. This methylation is associated with a reduction or elimination of Mu1 excision activity.
Mu Killer activity was originally identified when a high color background was crossed to a minimal Mutator line carrying a MuDR element at an unknown position. It was observed that this MuDR element became silenced in the progeny of this cross (Lisch and Freeling, 1994). In subsequent generations, the silencing effect was genetically separated from any full-length MuDR elements.
In the winter of 2000, a male carrying an active MuDR(p1) element was crossed to both a Mu permissive tester and MuK ears. The progeny of these crosses were analyzed by Southern blot. As expected, all (12 of 12 examined) of the progeny of the Mu permissive tester that carried MuDR(p1) had hypomethylated Mu1 elements. In contrast, of the progeny of the MuK plant that inherited MuDR(p1), 40 out of 78 (51.3%) had methylated Mu1 elements, as expected for the independent segregation of MuDR(p1) and MuK. All of these Mu1 methylated plants had been grown from weakly spotted or pale kernels carrying a full-length MuDR(p1). Pale kernels from this MuK ear were planted in the summer of 2001 and the resulting progeny were screened by DNA gel blot for the presence of MuDR. Although only half the progeny in this family were expected to carry MuK, none of those that carried MuDR(p1) exhibited signs of reactivation, demonstrating that even in the absence of MuK (due to segregation), silenced MuDR elements remain silent. Silenced MuDR(p1) elements also remained inactive when crossed to Mu permissive plants that did not contain MuDR (0/~1200 spotted kernels). Thus, MuK causes a heritable alteration of MuDR elements.
To test the heritability of MuK itself, several progeny plants from the above cross that lacked MuDR and that segregated for MuK were again crossed as females to males carrying MuDR(p1), and their progeny was analyzed by Southern blot. In one family that showed evidence of MuK activity (reduced excisions), 47.6% of the progeny that carried MuDR(p1) were methylated and inactive showing that MuK segregation was heritable (Table 1). In contrast, families derived from crosses between the same MuDR(p1) active plant and permissive testers resulted in all progeny with MuDR(p1) having hypomethylated Mu1 TIRs, demonstrating that MuDR was not inactivating prior to contact with the MuK ear.
Mu Killerís effect is not only on Mu1 TIRs. MuDR TIRs also become methylated in the presence of MuK. Thus, the inactivation of Mu1 is most likely a byproduct of MuDR silencing. Mu Killerís effect on MuDR is also not confined to the MuDR element at p1. MuK has been observed to silence single MuDR elements at other positions as well (Lisch and Freeling, 1994). Experiments are underway to determine if Mu Killer has the ability to silence complex Mu active lines currently used for transposon tagging. To date, MuK has been found not to cosegregate with any background MuDR deletion derivative.
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