In the 1982 Maize News Letter we presented a chart summarizing the interactions of the responding and controlling elements comprising previously studied two-unit mutable systems, as well as those of three new systems which arose in our high-loss studies. Several of the interactions had not been determined and were listed as "testing" or "to be confirmed." The missing diagnoses have now been ascertained and the complete set of interactions is given below where a plus signifies a positive and a minus sign a negative response. A positive interaction is evidenced either by breaks induced at the responding locus, followed by loss of the chromatin segment distal to the break, or by excision of the inhibiting Ds-like element from the affected locus, resulting in full or partial recovery of gene activity in a structurally intact chromosome. McClintock designated breakage as a Type I and excision of the regulatory element as a Type II event. Failure to find either breakage or excision would be scored as a negative response.
The Ds element used in our tests was proximal to Wx in the short arm of chromosome 9, and not between C-I and Wx as indicated in the pedigree provided by the Maize Stock Center. This Ds (called standard Ds) is not associated with any mutant phenotype and its interaction with Ac is demonstrated by Ac-induced breaks at Ds, resulting in the joint loss of the linked C-I and Wx markers. Phenotypically, there are colored, waxy (C wx) spots on a white, starchy (C-I Wx) background in endosperms of C wx/C wx/C-I Wx Ds; Ac constitution. Excision of Ds (Type 11 event) would not readily be detected with our Ds stock. It is only when transposition of Ds occurs and it is moved into or adjacent to a wild type structural gene, thereby inhibiting the activity of that locus and giving a recessive mutant phenotype, that excision of Ds from the inhibited locus is made evident by the recovery of full or partial gene activity in that cell and its descendants.
kernels lacking their specific controlling element. Mutability of a-mrh is induced by Mrh and not by Dt or En, mutability of a-standard by Dt and not by Mrh or En. and mutability of both a-m and a-mr by En but not by Mrh or Dt. The chart could be enlarged to include other responsive loci into which Ds has been inserted, but nothing new in principle would be gained.
The behavior of Ac2 is anomalous. When McClintock's Ac is tested against Ds in the C-I Wx Ds chromosome, a frequent loss of both C-I and Wx occurs as the consequence of Ac-induced breaks in the C-I Wx Ds chromosome (event Type I). Her Ac tested against bz2-m, which has a Ds-like element in or adjacent to a wild type Bz2 allele, causes the excision of Ds from Ds Bz2. We have not yet determined whether or not Ac causes excision of Ds (Type II event) from the C-I Wx Ds chromosome or breaks (Type I) in the bz2-m chromosome. However, since Ac2 also induces mutability of bz2-m (although differing from Ac in dosage effects, time of mutation and its restriction to specific tissues), it might be anticipated that Ac2 would cause breaks in the C-I Wx Ds chromosome. This was not the case. The frequency of C wx spots in C wx/C wx/C-I Wx Ds endosperms having two Ac2 was not discernibly different from that in similar endosperms with no Ac2. We concluded that Ds in the C-I Wx Ds chromosome differed from the Ds-like component in bz2-m, since the former gave a Type I response to Ac but not to Ac2. Both Ac and Ac2 induced Type II events at bz2-m, but they differed in that Ac induced excision of the Ds element of the wx-m-1 and c-m-1 alleles, while Ac2 was completely ineffective in eliciting a Type II response at either locus. Our tests prior to 1982 established that unlike Ac, Ac2 had no effect on Ds in the C-I Wx Ds chromosome, on mutability of wx-m-1 (= Ds Wx), on c-m-1 (= Ds C) nor on wx-m-9 (= Ac Wx). It seemed, therefore that the response of bz2-m to Ac and Ac2 was due to some unknown modification in the molecular structure of the Ds component of the bz2-m allele.
Some recently obtained data cast doubt on the validity of this conclusion. The response of bz2-m to increased doses of Ac2 is dramatic. No (or rare) Bz2 spots of aleurone color are found in kernels homozygous for bz2-m and possessing one Ac2, a low number occurs with two Ac2 and many mutations of bz2 to Bz2 are found with three doses of Ac2. Inasmuch as we had found (see our 1982 News Letter report) instances where two Ac2 elements were situated in juxtaposition in the same chromosome, we thought it of interest to see what the response of Ds in the C-I Wx Ds chromosome would be to four doses of Ac2. We had observed no detectable response of this Ds to two Ac2, but since the mutation rate of bz2-m was so dramatically increased in going from two to three doses of Ac2, it seemed possible that Ds in its standard position might exhibit a comparable response to higher numbers of Ac2.
Crosses of bz2-m/bz2-m; C-w wx/C-w wx; Ac2 Ac2/ac2 ear parent x Bz2/Bz2; C-I Wx Ds/C-I Wx Ds; ac2 pollen parent should give equal numbers of F1 endosperms with two genotypes: 1) bz2-m/bz2-m/Bz2; Ac2 Ac2/Ac2 Ac2/ac; C-w wx/C-w wx/C-I Wx Ds and (2) bz2-m/bz2-m/Bz2; ac2/ac2/ac2; C-w wx/C-w wx/C-I Wx Ds. The C-I allele was dominant to two doses of the weak C-w allele. Half of the kernels had four doses of Ac2 and one standard Ds, while the other half had no Ac2 and one Ds. A number of ears were obtained from this cross. The kernels on each ear fell into two distinct classes in a 1:1 ratio. One-half had a colorless aleurone with starch which stained blue with IKI. The majority of the kernels in this class had no colored-waxy (C wx) spots resulting from loss of the C-I Wx markers, but a low percent had a few-colored-waxy spots which were acribed to spontaneous loss. In sharp contrast to this group with no or low loss of the C-I Wx markers was the 50 percent of the kernels with a colorless-Wx background on Which there were many (literally hundreds) of colored-waxy spots. Clearly, frequent loss of C-I and Wx was occurring during endosperm development in this half of the F1 kernels, while the other half had no or a very low rate of loss. We knew from the pedigree of the parental plants that one-half of the kernels had four doses of Ac2 and the other half had none, but at this stage in our investigation we could not tell if the kernels with the high rate of C-I Wx loss also possessed four Ac2 elements with the other half having none. However, by testcrossing plants from kernels with a high rate of loss and plants from kernels with no loss of C-I Wx, we were able to conclusively demonstrate that all high rate kernels had four Ac2 while kernels with a low rate of loss had none. This demonstration was possible because the F1 plants were all heterozygous for bz2-m. If all bz2 kernels in a given testcross population had a stable bronze aleurone color, it was concluded that the two linked Ac2 were not carried by the tested plant. Conversely, if a testcrossed ear had equal numbers of bz2-mutable and bz2-stable kernels, the testcrossed parental plant was heterozygous for the two linked Ac2. Several score of plants coming from the two classes of endosperm were testcrossed, and without exception the kernels with a high rate of loss of the C-I Wx markers gave rise to plants which segregated for mutable and stable bronze-2 phenotypes in a 1:1 ratio. These plants were demonstrably heterozygous for Ac2. Plants derived from kernels with no or few losses of C-I and Wx had no Ac2 since no mutable bronze-2 kernels were found. These results challenge our earlier conclusion that Ds in its standard location does not respond to Ac2. It does respond, but only when four doses of Ac2 are present. We know that two or three doses of Ac2 induce Type II events (excision) at bz2-m but not in the wx-m-1 and c-m-1 mutable systems, and that increasing the dosage of Ac2 does not affect the mutation pattern of wx-m-9 as do extra copies of Ac.
In summary, breaks at Ds in its standard position are not induced by two doses of Ac2, while many breaks occur when the dosage of Ac2 is increased from two to four. This suggests that Ac2 is a weak variant of McClintock's Ac. Support for this tentative conclusion comes from the following observations. The wx-m-1 allele is a Wx allele with an inserted Ds which inhibits the activity of the Wx gene. Endosperms homozygous for wx-m-1, or having various combinations of wx-m-1 and recessive wx, have no Wx reversions if Ac is not present, but show many Wx spots upon the introduction of Ac. Since kernels with wx-m-1 and one to three doses of Ac2 have only wx starch in the endosperm, we concluded that Ac2 was unable to induce excision of the Ds element in wx-m-1. This observation strengthened our conclusion that Ac and Ac2 were too dissimilar for the one to be a mutant derivative of the other. However, in kernels with four doses of Ac2, standard Ds did undergo breakage, so a similar test was made of the effect of more Ac2 on-the wx-m-1 system. Waxy kernels from crosses of bz2-m/bz2-m; Ac2 Ac2/ac; wx/wx silks by Bz2/Bz2; ac2/ac2; wx-m-1/wx-m-1 pollen in the summer of 1982 gave endosperms of two different genotypes. They differed in that one class had four doses of Ac2 and the other class had none. All kernels are Bz2 phenotypically and have one dose of wx-m-1. Upon closely examining the endosperms from this cross, we found a few with an occasional, usually small, blue or lavender stained spot with IKI. Tests are presently being conducted to determine if all of the relatively infrequent kernels with rare blue or lavender staining cells are heterozygous for the two linked Ac2. Our prediction is that they are, and that wx-m-1 shows some response to four doses of Ac2 although it showed no response to one, two, or three doses. If our predictions are confirmed, a reasonable interpretation of our data might be that Ac2 is an anemic version of Ac, but we are hesitant in accepting this conclusion because the two differ in so many attributes. Whatever the outcome, it would appear that there is heterogeneity among the different Ds elements since they have dissimilar responses to the same activator. This saga will be continued in next year's News Letter.
M. M. Rhoades and Ellen Dempsey
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