A Second Hybrid Enzyme in MaizeAuthor(s): Drew SchwartzSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 51, No. 4 (Apr. 15, 1964), pp. 602-605Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/71974 .

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602 GENETICS: D. SCHWARTZ PROC. N. A. S.

more frequent (every Nd generations). Moreover, now the relative frequency of a is approximately NdIN which is essentially zero. In this process fluctuations of the

lnumber of a individuals of order Nd are observed. The difference between (ii) and (iii) is the magnitude of the mutatioll rate a2.

The limit processes exhibited in Theorem 4 are all of diffusion type. Theorem 5 describes a different sort of limit process valid as N -* X.

THEOREM 5. Let the conditions of Theorem 4 be satisfied. Let Pij(N) denote the transition probability mnatrix of X(t;N).

If a, = il N, a2(N) cx a2*, 0 < a2K <1, then

lim Pij(N) Pij* for all i, j = 0, 1,

and

E Pij*si - ((1 - a2*)S + a2*)eYl(i), j =o

that is, the limit process X*(t) of X (t;N) is a branching process with Poisson immi- gration. We interpret X*(t) as the number of individuals of the rare mutanlt a-type in an effectively infinite population of A-individuals.

The results of Theorem 4 and 5 are typical of a wide variety of limit processes coninected with the Markov chains of the transition probability matrices (3).

The theorems announced above testify to the rich structure the class of induced M:arkov chains (3) possesses. These results, further refinements, and extensions will be elaborated in other publications.

* Prepared under auspices of National Institutes of Health, grant GM 10452-OlAl. I Karlin, S., and J. McGregor, "Direct product branching processes and related induced markov

Chains, II. Calculation of rates of approach to homozygosity," to be published. 2 Kimura, M., "Some problems of stochastic processes in genetics," Ann. Math. Statist., 28,

882-901 (1957). 3Wright, S., "The genetical structure of populations," Ann. Eugenics, 15, 323-354 (1951).

A SECOND IIYBRID ENZYME IN MJAIZE*

BY DREW SCHWARTZ

DEPARTMNT OF BIOLOGY, WESTERN RESERVE UNIVERSITY, CLEVELAND, O1IO

Communicated by Boris Ephrussi, February 26, 1964

The common pH 7.5 esterases of maize, specified by the alleles ElJ, E1l, ald Els 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 formned by the El$ allele functioning bothb 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/EIE or E1F/1E hoimozygotes anid El1/ElF lheterozygotes. Tlhis esterase balnd is also seen in extracts of plants homozygous for four other less common alleles E1L E1u, E1T, and E1w, and has been referred to as the constant S band, The intensity

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VOL. 51, 1964 GENETICS: D. SCHWARTZ 603

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 analy- sis of the pH 7.5 esterase systenm. 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 7.3 esterase. ElF/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 zymlograms; i.e., both of the bands are present with the same relative intensity.

This interpretation has been confirmiied by recent studies. The gene which con- trols the synthesis of the constant S esterase is designated E3. A mlutanit 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 mlade 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 honmozygotes forms an esterase which occupies the position of the pH 7.5 SS esterase specified by the Els allele. For this reason, it had previously been referred to as the constant S esterase. E3s is the recently discovered miutant which in hoim-ozygous condition formis an esterase with a much slower migration rate. In the course of a 21/4-hr electrophoretic runl in starch gel at pH 8.5, the esterase formed in E3F/E3F homozygotes migrates about 8 mm toward the cathode, while the esterase formned in E3s/E3s homiiozygotes 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 result- ing from the action of the Els allele which could complicate the analysis.

Only a single esterase band is found at the positions of the E3 esterases in the E3 /E3F

and E3s/E3S honmozygotes. 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 positioni of E3F homozygotes; a second band is at the slow migrating position of the E3s homo- zygotes; the third band, found only in hetero- zygotes, and always accomiipanied 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 analy- sis is made on diploid tissue. These results suggest that the E3 esterases, like the El esterases, occur as dinmers. Analysis of the distribution of the E1 and E3 esterases in F2 progeny segregating for E1N and E1F as well

.t

;.

.. ..... ... . r.s.

s + v .S . +os+

*:: ..; :. ...< x.:

...... .... . e'F.:^.

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."

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604 GENETICS: D. SCHWARTZ PROC. N. A. S.

TABLE I

DISTRIBUTION OF GENOTYPES SEGREGATING IN F2 SEEDLING PROGENY OF THE CROSS EYFI/EYv, E3 F/E3F X E1N/E1N, E3S/E3S

Expected (without

Genotype Observed linkage)

E1N/E1N, E3s/E38 46 47 E1N/E1N, E3s/E3F 84 93 E1N/E1N, E3F/E3F 47 47 E1N/E1F, E3s/E3s 87 93 ETNI/EIF, E3S/E3S 185 186 E_N/E1F, E3F/E3F 87 93 E'/Ei1F , E3s/E3s 49 47 EiF/E11, E3s/E31'' 114 93 E1'/ElY, E3F/E3fi' 50 47

as E3S and E3F reveals that the two genes are not linked and segregate inde- pendently (Table 1).

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 umst 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 con- sider the possibility of some sort of close relationship between the two enzymnes 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. Ac- cording 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 mnodi- fies the esterase. If only the enzymes produced by the E1N and E1F alleles altered the charge of the esterase anid 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/ElF and E1I/E1N homozygotes should vary depending on the alleles present at the E3 locus. For example, the FF esterase band found in ElIE11', E3F/E3' plants should migrate faster than the esterase band in ElFjElF, E3s/E3s plants since the "precursor" enzyme produced by the E3F structur al gene would be imore positively charged than that of E3S. This is not the case. The position of the FF and NN bands are constant in E3s/E3s, E31IEj3 , and E3F/E3s genotypes, respectively.

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

The E1 and E3 esterase systems are the only systems thus far reported in maize where mutant genes have been shown to produce enzymiies with altered charge

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VOL. 51, 1964 BIOCHEMISTRY: SIEGEL, ROACH, AND POMEROY 605

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 vigorl may be a conmmon feature of maize enzymes.

Summary.-Two alleles of the E3 gene in rnaize specify electrophoretically sep- arable esterases. As is the case with the pH 7.5 esterases formed by the El gene, a hybrid esterase with an intermediate migration rate is found in the E3 heterozy- gotes. The enzyme specified by one of the E, alleles has a migration rate identical to that fornmed by one of the E, alleles.

* Research supported by National Science Foundation grant GB 302. 1 Schwartz, D., these PROCEEDINGS, 46, 1210 (1960).

PLASMA AMIINO ACID PAT77TERNS IN ALCOHOLISM: THE EFFECTS OF E7THANOL LOADING

BY FRANK L. SIEGEL,* MVIARY K. IROACH, AND LEON RI. POMEROY

CILAYTON FOUNDATION BIOCHEMICAL INSTITUTE, UNIVERSITY OF TEXAS, AUSTIN

Communicated by Roger J. Williams, January 31, 1964

Reports from several groups provide evidence which indicates that alcoholics have metabolic patterns which distinguish them from nonalcoholic subjects. Previous findings from this laboratory' include imbalances in the formed element composition of the blood of alcoholics, and of elevated blood levels of glucose, sodium, potassium, and calcium in this group. It was also reported that alcoholics have significantly elevated urinary excretion of hippuric acid, sodium, potassium, and chloride, and lowered excretion of creatinine. Application of these findings is complicated by ethnic group differences.2 More recently, we have presented a preliminary report' indicating imbalances of plasma amino acids in alcoholics, and group-distinguishing alteratiolls of the plasma amino acid pattern following acute ethanol ingestion by alcoholics and controls. Other laboratories have found that al- coholics, as a group, exhibit a defect of tryptophan metabolism,4 show reduced activ- ity of the sympathetic nervous system,5 tend to be vitamin B6- and B12-defi- cient,6'7 and to have low plasma levels of zinc8 and magnesium.9

Such metabolic imbalances may be regarded as the summation of two factors; the various metabolic and nutritional effects of chronic alcohol consumption, and conistitutional factors related to the etiology of alcoholism. Only when partition of these factors has been achieved can the iinportance of the genetic aspects be assessed. The present report on patterns of free amino acids in plasma describes the use of amino acid data obtained before and after ethanol loading to generate an hy- pothesis of a constitutional defect in alcoholism. The relative importance of en- vironmental stressers in the development of alcoholism is not denied, but it is suggested that constitutional factors may convey unusual susceptibility to such stress in alcoholism-prone individuals. Reed"0 and McClearn11 have documented evidence for genetic factors governing alcohol consumption in laboratory animals, and Williams"2 has indicated the potential v&Llue of approaching the problems of

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