We are interested in determining the relationship between the number of copies of Mutator or Mutator-related elements in the maize genome and the frequency of forward mutation at selected loci and the frequency of reversion from mutant to wild type at unstable alleles. For these studies we are using genes of the anthocyanin biosynthetic pathway. An interesting preliminary result concerns changes in the frequency of somatic reversion we see in the progeny of plants which differ in their Mu copy number
An unstable bz2 mutable (bz2-mu-1) with 25% of the surface covered in purple spots of 1-20 cells was recovered in 1982 from a cross of a purple aleurone Mutator line (kindly supplied by D. Robertson) as male onto bz2 tester in a hybrid W23/K55 background (kindly supplied by E. H. Coe, Jr.). The somatic reversion seen in the unstable bz2-mu-1 mutation is assumed to involve the excision of the Mu sequence from the allele restoring essentially wild type phenotype in revertant sectors. Because mutator insertion creates a short host sequence duplication, it is assumed that the unstable nature of the bz2-mu-1 mutation is due to the insertion of a copy of the mutator into the Bz2 gene in a position in which no permanent loss of gene function occurs after the element excises. For example, the element may have inserted into a noncoding region or other position where it eliminates transcription or prevents proper processing, and upon excision the duplicated sequence does not interfere with the gene's expression. The mutant was backcrossed to the hybrid tester in the greenhouse, and progeny kernels, each with approximately 25% of the total surface area occupied by small revertant sectors encompassing 1-20 cells of the aleurone, were planted in the 1983 summer crop. At the 8-leaf stage a lower leaf was removed, DNA prepared, and slot blot hybridization carried out by M. Fromm using a cloned Mutator (Mu) element as the radioactive probe to DNA samples from each of 30 plants. The hybridization results clearly showed that there were plants with high, medium and low copies of Mu or sequences cross-hybridizing with mutator. There was approximately a 10-fold difference in copy number between the high and low classes.
Each classified plant was then testcrossed by the hybrid bz2 tester, crossed onto several hybrid tester ears, and the second ear self-pollinated. In these progeny ears we have scored the occurrence of revertant sectors. In those kernels with revertant sectors, the spots are still about 1-20 cells in size. The expectation, barring any effects of mutator copy number, is that in each outcross ear 50% of the kernels will have spots because they are bz2-mu-1/bz2 and 50% of the kernels will be colorless (bz2). In a self-pollination, 75% of the kernels are expected to have spots: 1/4 bz2-mu-1/bz2-mu-1, 1/2 bz2-mu-1/bz2, 1/4 bz2/bz2. The table summarizes the observed distribution of kernels with somatic sectors for eight plants in the experiment:
With outcrosses, the frequency of spotted kernels was independent of whether the mutator plant was the ear or pollen parent. In self-pollinated ears with the expected number of spotted kernels, it is interesting to note that approximately 1/3 of the spotted kernels were 50% or more occupied by somatic sectors and 2/3 of the kernels were 10-30% occupied by somatic sectors, suggesting that the heavily spotted kernels are the homozygotes and that each copy of bz2-mu-1 can revert independently. Tests of these suppositions are underway.
The unexpected observation is that, although self-pollinated ears of low copy number parents show 75% kernels with spots, the outcross ears in two families fail to have as many revertant kernels as would be expected from the segregation of the mutable allele. Naively we can propose that in the testcross either the number of Mu copies or some other factor was lost or diluted out so that reversion becomes less frequent (or very much later in development). Conversely, in high copy number parents self-pollination, which could increase the copy number of Mu or of another factor influencing Mu behavior, drastically reduces the number of kernels with spots, although in testcrosses 50% of the kernels are spotted. This result suggests that a high copy number of Mu may also prevent somatic reversion.
It is possible that Mu encodes both a transposase which can become rate-limiting at low Mu copy number, as well as a repressor (of transposition or of transposase gene transcription) which at a high Mu copy number effectively prevents excision of the elements. In such a model only lines carrying an intermediate number of Mu copies will have an active mutator system viewed from the perspective of excision from known loci. Indeed the medium copy number parents in this experiment demonstrated expected segregation of spotted kernels in both testcrosses and in self-pollination. Mutator activity in transposition and insertion at other sites may or may not parallel the frequency of Mu excision from a known mutable allele. Tests are underway to determine whether mutation frequency, a measure of transposition and insertion, is affected to the same extent and in a manner parallel to excision frequency in somatic tissue in lines with varying numbers of Mu sequences. We are also testing whether the quiescent bz2-mu-1 allele in low copy number self ears can be activated by crossing in Mu copies from a bz2 kernel with a high number of copies of Mu, and whether the quiescent bz2-mu-1 alleles in self-pollinated high copy number ears can be activated by outcrossing the progeny plants to lines lacking mutator activity, a cross which should dilute the active copies of the Mu sequence.
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