In last year's newsletter (MGNL 55:8) we presented evidence that the wx controlling element mutants m-1, m-6, B3 and possibly m-8 and B4 lie within the limits of the Wx structural gene. The evidence given for the Mp mutation in B3, originally obtained from Oliver Nelson, was based on the observation that this mutant produces an altered, inactive Wx protein. However, additional B3 material, subsequently obtained from the Coop., gave completely different results. It turns out that B3 had been established in two different backgrounds, W22 and M14, and that the mutant maintained in each background is different. The B3 we originally reported on, which produces the normal amount of an altered Wx protein, is in the W22 background and recent analysis shows that it does not have any Mp transposing activity. In contrast, the B3 allele that is in the M14 background does have MR activity but produces very little Wx protein. These alleles will be referred to as B3-W (W22) and B3-M (M14). B3-M was the allele that Nelson used in his mapping studies (MGNL 50:109) The B3 pedigree, provided by Nelson, shows that the B3-M allele had been backcrossed into M14 through four generations. The B3-W allele came from a cross of B3-M to W22 and two subsequent backcrosses to W22 followed by two generations of self-pollination. We have shown that the differences in Wx protein level and Mp activity between B3-W and B3-M are not due to the differences in background. The evidence indicates that B3-W is a stable germinal derivative of B3-M. In addition, the mutable B3-M allele seems to undergo autonomous changes in the endosperm to produce both an active and inactive Wx protein.
Since Nelson used the B3-M and not the B3-W allele in his mapping studies, it is important to establish that the B3-M mutant, like B3-W, involves an alteration within the structural gene limits. A pollen recombinational analysis was performed on the two B3 alleles according to the methods of Nelson (Genetics 60:507). The data are given in Tables 1 and 2. It is concluded that there is no detectable recombination between the two B3 alleles. Hence, the Mp element of B3-M is at or very close to the site of the B3-W mutation and most probably lies within the structural gene limits. The evidence presented below that the B3-M mutant can give rise to an altered Wx protein also indicates that Mp lies within the structural gene.
The Mp activity of B3-M is shown by the somatic reversion to Wx in endosperm heterozygous for B3-M and the wx Ds alleles m-1, m-6 or B4. Some autonomous somatic reversions to Wx are also observed in B3-M/wx/wx endosperm. Whereas the Mp element of B3-M is as active as Ac in inducing endosperm reversions in the wx Ds alleles, B3-M is only very slightly active in inducing its own reversions. Each B3-M/wx/wx endosperm has only a few small Wx sectors containing only one or a few cells, as seen with IKI staining.
The Wx protein can be extracted from endosperm starch granules and visualized in SDS polyacrylamide gels following electrophoresis and staining (MGNL 55:9). As previously reported, the Wx protein from B3-W is produced in the normal amount. The Wx protein from B3-M, however, is usually present in a greatly reduced amount (roughly 5% the one-dose B3-W level) and decreases with increasing dosage of the B3-M gene or with the presence of additional Ac's. When individual kernels are screened for their Wx protein, about 10% are exceptional and show an increased amount of Wx protein above that seen in the rest of the population. The degree of the increase varies, and although it is usually small, it can approach the one-dose level seen for B3-W or Wx. Because mature kernels from these same crosses do not show an increase in blue staining starch with IKI, most of the Wx protein produced by these exceptional B3-M kernels must be inactive.
It also appears that, even in the non-exceptional kernels, most of the Wx protein present is enzymatically inactive. This is based on a comparison of the amount of Wx protein and blue staining starch produced in the endosperm by B3-M to that produced by the wx Ds mutant m-6 in response to B3-M. In the absence of Mp or Ac, m-6 is stable and produces neither reversions nor Wx protein. When one Ac is present, numerous reversions are seen in the endosperm and a Wx protein is produced that appears about 1,000d larger than the standard 60,000d Wx protein in migration comparisons in Laemmli SDS gels. In m-6/m-6/B3-M heterozygotes two Wx proteins are made, the standard sized B3-M protein and the apparently larger m-6 protein. In silver stained gels the amount of B3-M Wx protein is only slightly less than the amount of m-6 Wx protein. Based solely on the protein ratios, one would predict that B3-M should give rise to the same degree of Wx reversion as m-6 does in response to B3-M or Ac. However, the amount of blue staining starch in B3-M/wx/wx endosperm is at least 10-fold less than that seen in m-6/m-6/B3-M or m-6/m-6/wx ; Ac endosperm. Such disparity between the amount of Wx protein and blue staining starch produced by B3-M suggests that nearly all of the Wx protein produced is enzymatically inactive. Though some active protein must be produced, because a few small Wx sectors occur in B3-M kernels, this small amount of protein would hardly be detectable.
It is proposed that B3-M is undergoing the normal amount of autonomous transposition expected of an Mp mutant but that an inactive, altered Wx protein is the result. It is only rarely that a "mistake" is made and an active Wx protein is produced. Preliminary isoelectric focusing analysis suggests that the proteins produced by B3-M and B3-W have the same isoelectric point.
The production of an altered Wx protein by m-6 reversions provides further evidence that the Ds element in m-6 lies within the structural gene limits.
Craig Echt and Drew Schwartz
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