PNAS 51(4): 602-605 (Apr 15, 1964)
Communicated by Boris Ephrussi, February 6, 1964

* Research supported by National Science Foundation grant GB 302.

The common pH 7.5 esterases of maize, specified by the alleles E1F, E1N, and E1S occupy five positions in zymograms developed after starch gel electrophoresis. The position closest to the point of origin is occupied by the SS band which is formed by the E1S allele functioning both in homozygous or heterozygous plants. However, as was reported in an earlier publication,1 an esterase band is found in the SS position even in extracts of plants which lack the E1S allele, such as E1N/E1N or E1F/E1F homozygotes and E1N/E1F heterozygotes. This esterase band is also seen in extracts of plants homozygous for four other less common alleles, E1L, E1R, E1T, and E1W, and has been referred to as the constant S bind, The intensity of this band relative to the bands of the pH 7.5 esterases is variable, but it is always weaker than the latter. The constant S band has in the past complicated the analysis of the pH 7.5 esterase system. This esterase was not observed to hybridize with nor segregate from the other pH 7.5 esterases, and was presumed to be specified by another locus, other than the E1 gene which specified the pH 7.5 esterase. E1F/E1F plants show two bands, an intense band at the FF position and a weak band at the SS position. If such a plant is self-pollinated, all of the progeny give identical parent-like zymograms; i.e., both of the bands are present with the same relative intensity.

Fig. 1.—Zymograms of extracts from homozygous and heterozygous E3 genotypes. The arrows designate the positions of the E3 esterases. The faster-migrating heavy bands are the pH 7.5 esterases. The cathode is at the bottom and the origin is indicated by an ''O.''

This interpretation has been confirmed by recent studies. The gene which controls the synthesis of the constant S esterase is designated E3. A mutant allele of this gene has recently been found in our laboratory. The mutant is characterized by the synthesis of an esterase with an altered migration rate. The electrophoretic and experimental procedures used in this study are described in the earlier paper.1 The electrophoretic analysis was made on seedling material. The two alleles at this locus have been labeled E3F and E3S. E3F is the allele carried by most of the lines of maize which have been investigated and in homozygotes forms an esterase which occupies the position of the pH 7.5 SS esterase specified by the E1S allele. For this reason, it had previously been referred to as the constant S esterase. E3S is the recently discovered mutant which in homozygous condition forms an esterase with a much slower migration rate. In the course of a 2 1/4-hr electrophoretic run in starch gel at pH 8.5, the esterase formed in E3F/E3F homozygotes migrates about 8 mm toward the cathode, while the esterase formed in E3S/E3S homozygotes shows only a very slight migration, about 1 mm from the origin in the same direction. In these studies we have used plants which carry only the E1N and/or E1F alleles so that there would be no enzyme at the SS position resulting from the action of the E1S allele which could complicate the analysis.

Only a single esterase band is found at the positions of the E3 esterases in the E3F/E3F and E3S/E3S homozygotes. However, as was found to be the case with the E1 heterozygotes, E3F/E3S heterozygotes form three esterase bands. One band is at the faster-migrating position of E3F homozygotes; a second band is at the slow migrating position of the E3S homozygotes; the third band, found only in heterozygotes, and always accompanied by the other two, has an intermediate migration rate (Fig. 1). The third or hybrid band is more intense than either of the parental bands if the analysis is made on diploid tissue. These results suggest that the E3 esterases, like the E1 esterases, occur as dimers. Analysis of the distribution of the E1 and E3 esterases in F2 progeny segregating for E1N and E1F as well as E3S and E3F reveals that the two genes are not linked and segregate independently (Table 1).

E1F/E1F, E3F/E3F × E1N/E1N, E3S/E3S

Genotype Observed Expected
E1N/E1N, E3S/E3S 46 47
E1N/E1N, E3S/E3F 84 93
E1N/E1N, E3F/E3F 47 47
E1N/E1F, E3S/E3S 87 93
E1N/E1F, E3S/E3S 185 186
E1N/E1F, E3F/E3S 87 93
E1F/E1F, E3S/E3S 49 47
E1F/E1F, E3S/E3F 114 93
E1F/E1F, E3F/E3F 50 47

As was mentioned at the outset, the occurrence of an esterase band at the pH 7.5 SS esterase position in all genotypes had in the past introduced a complication in our analysis of the pH 7.5 esterase system. It had been suggested that this esterase must be specified by a gene other than E1. However, the fact that this esterase banded out at precisely the same position as the SS band forced us to consider the possibility of some sort of close relationship between the two enzymes rather than of a fortuitous coincidence in migration rates. One scheme which had to be reckoned with was that E1 is not the structural gene for the pH 7.5 esterase. According to this alternative, the esterase is specified by some other locus, say E3, and has the migration rate of SS. The E1 gene would form an enzyme which modifies the esterase. If only the enzymes produced by the E1N and E1F alleles altered the charge of the esterase and not all of the "precursor" esterase molecules were modified by the E1 gene, some esterase would always be found at the SS position regardless of the E1 genotype. The finding of a mutant allele at the E3 locus negates this possibility and shows that the E3 and E1 esterases are under the control of two different structural genes. If the E1 gene simply modified the esterase specified by the E3 gene, then the position of the FF and NN bands found, respectively, in E1F/E1F and E1N/E1N homozygotes should vary depending on the alleles present at the E3 locus. For example, the FF esterase band found in E1F/E1F, E3F/E3F plants should migrate faster than the esterase band in E1F/E1F, E3F/E3F plants since the "precursor" enzyme produced by the E3F structural gene would be more positively charged than that of E3S. This is not the case. The position of the FF and NN bands are constant in E3S/E3S, E3F/E3F and E3F/E3S genotypes, respectively.

E1 and E3 are probably not duplicate genes specifying the same enzyme but situated at different loci. If this were the case, hybrid enzymes should be formed between the products of the E1 and E3 genes even when each is present in homozygous condition. As was shown above, this is not so. Hybrid enzymes occur only in E1 heterozygotes and E1 heterozygotes, not in the homozygotes. Of course, one gene may have arisen from the other by duplication and become altered by mutation to such an extent that it now specifies a structurally different esterase, but these can no longer be considered duplicate genes.

E1 and E3 esterase systems are the only systems thus far reported in maize where mutant genes have been shown to produce enzymes with altered charge rather than to cause the absence of the wild-type enzyme. It is of interest that in both of these cases hybrid enzymes are formed in heterozygotes. Dimerization which has been proposed as the basis for hybrid vigor1 may be a common feature of maize enzymes.

Summary.—Two alleles of the E3 gene in maize specify electrophoretically separable esterases. As is the case with the pH 7.5 esterases formed by the E1 gene, a hybrid esterase with an intermediate migration rate is found in the E3 heterozygotes. The enzyme specified by one of the E3 alleles has a migration rate identical to that formed by one of the E1 alleles.

1 Schwartz, D., these PROCEEDINGS, 46, 1210 (1960).