An intergenic alcohol dehydrogenase isozyme in sunflowers

The isozymes of alcohol dehydrogenase (ADH; E.C. 1.1.1.1) in wild and cultivated sunflower (Helianthus annuus) seeds can be resolved electrophoretically into 12 bands. The slowest- and probably the fastest-migrating sets of three are allozymic products of two genes, Adh ₁ and Adh ₂ , each having two alleles, F (for fast) and S (for slow). Evidence from dissociation-recombination experiments utilizing bands excised from starch gels indicates that an intermediately-migrating isozyme is a dimeric intergenic product consisting of ADH-1F and ADH-2S subunits. The hybrid isozyme was unstable in vitro in that its monomers spontaneously dissociated and recombined to produce ADH-1FF and ADH-2SS isozymes. The molecular weights of the hybrid as well as the parental isozymes were estimated at approximately 98,000.Supported by a Graduate School Research grant of the University of Kansas and by NSF grant GB-35853.     

Dissociation-recombination of intergenic sunflower alcohol dehydrogenase isozymes and relative isozyme activities

Two unlinked genes, Adh ₁ and Adh ₂, control the production of alcohol dehydrogenase (ADH) in seeds of the annual sunflower (Helianthus annuus). Each gene is polymorphic, having F and S alleles. Starch gel electrophoretic zymograms of the four possible double homozygotes have three bands, representing two homodimers and an intermediately migrating intergenic isozyme. Zymograms of double heterozygotes consist of nine bands produced by ten isozymes: six intragenics and four intergenics, two of which are coincident. Results of dissociation-recombination (D-R) experiments are reported which demonstrate the subunit composition of the intergenic isozymes, thus supporting the relationships suggested by genetic studies. Densitometric tracings of the zymogram of a cleared gel and measurements of activities of homodimer isozymes eluted from gels following D-R of an intergenic isozyme showed that the Adh ₂ isozymes were more than twice as active as those of Adh ₁. Measurements of activities of crude extracts from the four possible double homozygous genotypes indicated that the seeds of the genotype Adh ₁ ^F /Adh ₁ ^F , Adh ₂ ^S /Adh ₂ ^S produced more activity than the other three. This genotype is the most common one found in wild and cultivated stocks. Isozymes eluted following electrophoresis of the same extracts had averages of 19%, 70%, and 11% of total activity contributed by the Adh ₁, Adh₂, and intergenic isozymes, respectively. A simple but efficient method of isozyme elution from starch gels is described which resulted in nearly full expected recovery (approximately 46%) of the ADH activity in the applied sample.Supported by Graduate School and BioMed grants and by NSF Grant GB35853.     

Comparisons of Relative Activities of Maize Adh(1) Alleles in Heterozygotes-analyses at the Protein (Crm) Level

The method of high-resolution electrophoresis was employed to compare the relative amounts of enzyme produced by the Adh₁^F and Adh₁^S alleles in heterozygotes at different stages of development. The results are in complete agreement with those obtained from enzymatic analyses and support the competition hypothesis for the regulation of the alcohol dehydrogenase gene.     

Sunflower alcohol dehydrogenase: ADH1 genetics and dissociation-recombination

The composite alcohol dehydrogenase zymogram of sunflowers, Helianthus annuus, consists of 12 distinct bands. Genetic studies suggest that the slowest-moving three bands are allozymic dimers. They are controlled by a gene designated Adh ₁ having two codominant alleles, Adh ₁ ^F and Adh ₁ ^S . The heterozygote produces three bands as expected with a dimer molecule, while the homozygotes produce but one band each, consisting of FF or SS homodimers. The genetic evidence is supported by dissociation-recombination experiments in which the homodimers were separated and allowed to rejoin as parental homodimers and the hybrid heterodimer. Adh ₁ ^FS was found in only three of 422 cultivar seeds of one collection out of about 70 (over 6000 individual seeds tested) and was seen only infrequently in the seven wild collections examined. Adh ₁ ^SS has never been found in the cultivar collections studied and but rarely in the wild populations sampled.     

Association and the Adh locus with a lethal factor (l(2) Stm) in Drosophila melanogaster

Keeping Drosophila cultures at 28 C results in elimination of all minor multiple ADH bands, thought to be due to conformational change. Thus in diploid and triploid adults heterozygous for the Adh ^F and Adh ^Salleles, relative staining intensities are found for the three bands which were in conformity with the assumption that both alleles are equally expressed. Among all polymorphic strains derived from natural Central European and Mediterranean populations, the strain ⁺Tüb is unique in that its Adh ^Fallele is closely linked to a new recessive lethal factor, named 1(2)Stm. All Adh ^F 1/Adh^F 1 pupae are unable to emerge, and die. The lethal effect is obvious 50 hr earlier by retarded eye, bristle, and body wall pigmentation. Although all pupae of the phenotype F die, Adh ^F allele frequency scarcely seems to be lowered in this natural population.     

Dimerization of multiple maize ADHs studied in vivo and in vitro

Anaerobically induced primary roots simultaneously express two alcohol dehydrogenase (Adh) genes which specify three types of electrophoretically separable dimers: Set I, II, and III ADH. The S inbred line yields a particular activity ratio among these three sets. By use of an Adh ₁ ^null mutant allele and in vitro chemical dissociation and reassociation of ADH dimers, these studies extrapolate from an ADH activity ratio to the actual ratio of ADH protein. Conclusions are that (1) ADH₁ and ADH₂ promoters dimerize randomly in vivo and in vitro, (2) the heterodimeric isozyme (Set II) is approximately the enzymological sum of its subunits under these assay conditions, and (3) ADH-2 subunits are from 10 to 20% as active as ADH₁ subunits under these assay conditions. These conclusions imply that the unlinked Adh genes are coordinately regulated and reconfirm the two-gene-three-dimer model for the maize ADH isozymes.     

Mutations of the Adh1 gene in maize following infection with barley stripe mosaic virus

Summary Mutations at the Adh1 locus in maize were selected from plants infected with barley stripe mosaic virus (BSMV). Pollen from the infected inbred line 1s2p, which is homozygous for Adh1-S (abbreviated S), Adh2-P, c and r was treated with allyl alcohol and applied to silks of a tester stock homozygous for Adh1-F, Adh2-N, C and R. From these pollinations 356 kernels arose on the F₁ ears. Of these eight showed no activity of the S allele in scutellar samples while two exhibited low levels. Five of the putative mutant kernels germinated and two of these contained the contamination markers Adh2-P, c and r. The newly arisen mutations were designated S5446 and S5453. S5453 exhibited an abnormally low level of ADH activity in the F₁ scutellum. In the F₂ generation the mutant reverted at a high frequency with only about 5% of the S5453 alleles expressing low levels. DNA blotting and hybridization analyses showed no alterations in the restriction patterns of S5453 when compared to the progenitor S allele. S5446 which exhibited no ADH activity in the F₁ scutellum is unstable in the pollen; reversion frequencies approaching 10^-2 were observed in samples from some plants. Restriction digestion patterns of DNA from this mutant revealed the presence of a 3.3 kb insertion at Adh. The insert does not appear to contain sequences homologous to the BSMV genome but rigorous analyses remain to be carried out. It is hypothesized that BSMV infection may mobilize endogenous but dormant transposable elements in maize.     

Association of Chromosome and Enzyme Polymorphisms in Natural and Cage Populations of DROSOPHILA MELANOGASTER

The frequencies of a polymorphic inversion, In(2L)t, and of Adh and αGpdh alleles were analyzed in three natural populations of Drosophila melanogaster from Japan. Significant positive correlations between the frequencies of In(2L)t and Adh^S or αGpdh^F were detected due to tight linkage. An analysis of correlation with latitude showed that the negative cline of Adh^S frequency could be explained entirely by its linkage with In(2L)t; the frequency of Adh^S on the standard chromosome did not show a latitudinal cline. To the contrary, the cline of αGpdh^F frequency itself was positive, and its linkage with In(2L)t makes the positive cline unclear. These results suggest that the two allozymes themselves respond to latitudinal natural selection in different ways. When these populations were transferred to laboratory cages and maintained for a long time, they lost the chromosomal polymorphism but retained stable enzyme polymorphisms, although allele frequencies in the cage were not the same as in nature. The frequencies of Adh and αGpdh alleles were close to those in earlier cage populations of the same geographical origin.     

Partial Correction of Structural Defects in Alcohol Dehydrogenase through Interallelic Complementation in Drosophila melanogaster

Alcohol dehydrogenases (ADH) from the F₁ progeny of all pairwise crosses between 12 null-activity mutants and crosses between these mutants and four active variants, ADHⁿ⁵ ADH^F, ADH^D and ADH^S, were analyzed for the presence of active or inactive heterodimers. Gels were stained for ADH enzyme activity, and protein blots of duplicate gels were probed with ADH-specific antibody to detect cross-reacting material. Crosses between the three major electrophoretic variants. ADH^F, ADH^S and ADH^D, all produced active heterodimers. Four mutant proteins (ADHⁿ², ADHⁿ⁴, ADHⁿ¹⁰ and ADHⁿ¹³) did not form heterodimers with any other ADH subunit tested. Of the 28 crosses involving the remaining null activity mutants, 22 produce heterodimers. Twelve of these exhibit partial restoration of enzyme activity. In five cases of active heterodimers from null-activity crosses, Adhⁿ¹¹ supplied one of the subunits. In two crosses involving the active variant ADH^D, the null activity mutant subunits (ADHⁿ⁸ and ADHⁿ³) destabilized the heterodimer sufficiently to cause inactivation of the ADH^D subunit. In the cross between Adh^F and Adhⁿ³, the activity of the ADH^F subunit was also greatly reduced in association with the ADHⁿ³ subunit. Two crosses (Adhⁿ¹ x Adhⁿ¹¹ and Adhⁿ⁵ x Adhⁿ¹²) result in partial restoration of one of the homodimeric proteins (ADH ⁿ¹ and ADHⁿ¹², respectively), as well as forming active heterodimers.     

Mimicry of isozyme adaptive advantage by gene association I. Relationship between Adh genotypes and body dimension in Drosophila cage populations: A multivariate analysis

Experiments are described that are to prove that the apparent selective responses at the Adh locus in Drosophila melanogaster are not independent from its genetic background and from the variation at the gene pool level brought about by the changes of selection pressure. The dynamics of allozyme frequencies were observed at the Adh locus, of five metric traits and of reproductive fitness in two synthetic populations of Drosophila melanogaster originated from the same cross between Canton and Oregon strains, homozygotes for different Adh alleles and reared at different temperature (25 °C and 28 °C), until all above mentioned characters showed no more variations. The results obtained by univariate and multivariate statistical analyses can be summarized as follows:

- Adh ^s allele frequency keeps high and reaches very similar values in spite of the different environmental temperatures, whose selective effects at the Adh locus are therefore unlikely;

- both populations evolve toward the stabilization of Adh frequencies and other characters with a process strictly dependent on the permanence of coadapted blocks of genes which were contributed to the F₂ generation by the parental Canton and which are identified phenotypically by the association of Adh ^s/s with short wing;

- at the stabilization point the flies classified on the basis of their Adh genotype exhibit different shapes, namely their metric phenotypes can be discriminated considering all the respective traits together by means of multivariate analyses.

Owing to the presence in the initial populations of heterosis and epistatic interactions between loci, the observed differences between Adh genotype groups should represent the outcome of selection upon coadapted blocks of genes rather than on individual loci. Therefore, these results are argued as further evidence that each Adh genotype can be associated to different gene arrangements and its adaptive value cannot be isolated from that of its genetic background.     

A long-term study on interactions between the Adh and αGpdh allozyme polymorphisms and the chromosomal inversion In(2L)t in a seminatural population of D. melanogaster

The Adh and αGpdh allozyme loci (both located on the second chromosome) showed considerable fluctuations in allele frequencies in a seminatural population of Drosophila melanogaster during 1972–97. Both long-term and short-term fluctuations were observed. The short-term fluctuations occurred within almost all years and comparison of allele frequencies between winters and summers showed significantly higher Adh^S (P < 0.001) and αGpdh^F (P < 0.01) allele frequencies in summers. Frequencies of these alleles were significantly positively correlated with environmental temperature, suggesting the adaptive significance of these allozyme polymorphisms. Frequency changes of the Odh locus (located on the third chromosome) showed no seasonal pattern and were not correlated with environmental temperature. Almost all short-term and long-term increases in Adh^S frequency were accompanied by a corresponding decrease in αGpdh^S frequency (r = –0.82, P < 0.001) and vice versa. Further analysis showed that gametic disequilibria between the Adh and αGpdh loci, which frequently occurred, were due to the presence of inversion In(2L)t located on the same chromosome arm and In(2L)t frequencies were positively correlated with environmental temperature. Gametic disequilibria between Adh and Odh and between Odh and αGpdh were hardly observed. Because In(2L)t is exclusively associated with the Adh^S/αGpdh^F allele combination, the observed correlated response in Adh/αGpdh allele frequencies is (at least partly) explained by hitchhiking effects with In(2L)t. This means that the adaptive value of the allozyme polymorphisms has been overestimated by ignoring In(2L)t polymorphism. Fluctuations in Adh allele frequencies are fully explained by selection on In(2L)t polymorphism, whereas we have shown that αGpdh frequency fluctuations are only partly explained by chromosomal hitchhiking, indicating the presence of selective differences among αGpdh genotypes in relation with temperature and independent of In(2L)t. Frequency fluctuations of αGpdh and In(2L)t are consistent with their latitudinal distributions, assuming that temperature is the main environmental factor varying with latitude that causes directly or indirectly these frequency distributions. However, the results of the tropical greenhouse population show no correlation of Adh (independent of In(2L)t) and Odh allele frequencies with environmental temperature, which may indicate that the latitudinal distribution in allele frequencies for these loci is not the result of selection on the F/S polymorphism in a direct way.     

Regulation of the activity of alcohol dehydrogenase isozymes during the development of the maize kernel

Summary The relative activities of alcohol dehydrogenase isozymes have been studied during the development of the endosperm and scutellum of heterozygous Adh ₁ ^F /Adh ₁ ^S maize kernels. The products of the Adh ₁ ^F allele are found earlier than the products of the Adh ₁ ^S allele in both the scutellum and the endosperm. A second gene (Adh _r )which controlsthe activity level of ADH is active in the scutellum only. The Adh _r ^N allele specifies increase in the relative activity of the Adh ₁ ^S products from 26 to 38 days after pollination. This increase is prevented by the Adh _r ^L allele which is dominant. These results ar discussed on the basis of the limited factor hypothesis proposed recently by Schwartz (1971) for the regulation of the Adh ₁gene in maize.     

The <Emphasis Type="Italic">Adh</Emphasis> locus and adaptation of <Emphasis Type="Italic">cn</Emphasis> and <Emphasis Type="Italic">vg</Emphasis> mutants in experimental populations of <Emphasis Type="Italic">Drosophila melanogaster MEIG</Emphasis>

A complex study on the adaptation of cn and vn mutants and the allozymes of alcoholdehydrogenase (ADH) was carried out in initially pure lines, and their panmixia populations during exchange of the mutant genotype with that of wild-type flies (C-S) and D) through saturating crossings. The relative adaptation of the genotypes was estimated by their effect on reproductive efficiency in the experimentally obtained population. Fecundity, lifespan, and the resistance of the studied genotypes to hyperthermia were investigated individually. It was shown that the high level of adaptation of the cn mutants and the low level of adaptation of the vg mutants was correlated with the presence of different ADH allozymes. In the studied population, the F-allozyme of ADH accompanied the vg mutation, while the S-allozyme of the enzyme was detected in cn mutants. Saturating crossings of C-S(Adh ^S)×vg(Adh ^F) and D(Adh ^F) × cn(Adh ^S), along with the parallel determination of the allele composition of the Adh locus, demonstrated that the complete substitution of the F-allozyme of ADH in the vg mutants by the S-allozyme in D flies, as well as the substitution of the S-allozyme of ADH in the cn mutants by the F-allozyme in D flies was realized only after the 15th–20th backcrosses. These results favor the coadaptation of cn and vg marker genes with alleles of the Adh locus and indicate the important role of the latter in the adaptation of genotypes. In the studied population, selection acted primarily against the vg mutants, which were inferior to the cn mutants, and heterozygote genotypes in indices of the main adaptation components.     

Genetic relationships between the multiple alcohol dehydrogenases of maize

There are three electrophoretically separable sets of alcohol dehydrogenase isozymes in maize. Previous work has shown that two of these isozymes (Sets I and II) share a subunit in common, since mutations in one of the Adh genes, Adh ₁, alter both isozymes. A mutation in the second Adh gene, Adh ₂, has now been induced and recovered. This mutant allele also alters two of the three isozymes—Sets III and II. Adh ₁ and Adh ₂ appear to segregate independently. Gel filtration data show that all ADH isozymes are indistinguishable in size. These findings support the hypothesis that the two Adh genes specify promoters which homo- and heterodimerize, yielding three types of ADH isozymes.This research was supported by National Science Foundation Grant GB 25594. M.F. is a recipient of Public Health Service Genetics Training Grant GM 82-12.     

Alcohol dehydrogenase allozymes in the safflower genus Carthamus L.

The ADH allozyme pattern was tested in seeds of 1553 varieties of the world collection of safflower (Carthamus tinctorius L.) and 36 collections belonging to 14 wild species of the genus Carthamus L. with different chromosome numbers (n=10, 11, 12, 22, and 32). Two genes, Adh ₁ and Adh ₂, have been identified. The Adh ₁ locus controls the allozyme bands found in the faster-moving anodal zone, and the Adh ₂ gene controls the cathodal band. A third group of bands which migrates slowly toward the anode and stains weakly is probably interaction products of the two genes. Two codominant alleles Adh ₁ ^S and Adh ₁ ^F , specifying allozymes with different migration rates in the fast-moving anodal zone, were found in cultivated safflower. The frequency of the Adh ₁ ^F allele was very low. A third homologous allele, Adh ₁ ^T , was present only in the polyploid wild species. The Adh ₂ was stable, without any variation in migration rate. In addition to the variation in migration rates, there was also variation in activity levels of the products of both the Adh ₁ and Adh ₂ genes. The contribution of this study to our understanding of the origin of the polyploid species C. lanatus, C. baeticus, and C. turkestanicus is discussed.This research has been financed in part by a grant made to A. Ashri by the U.S. Department of Agriculture, Agricultural Research Service, authorized by P.L. 480, Project No. A10-CR-18, Grant No. FG-Is-234.Based in part on a thesis submitted by M. P. to the Faculty of Agriculture, The Hebrew University, Rehovot, in partial fulfillment of the requirements for the M.Sc. degree.Graduate Student, Faculty of Agriculture.     

Analysis of genetic variation affecting the relative activities of fast and slow ADH dimers in Drosophila melanogaster heterozygotes

When Adh ^F /Adh ^S heterozygote homogenates are stained after electrophoresis, considerable variation is observed in the activity ratio of the FF dimer to the SS dimer. Two Adh ^S strains showed a sharp, consistent difference when crossed to a common Adh ^F strain. Optical scanning and genetic analysis confirmed that this difference originates close to the Adh locus. Since the morphs varied concordantly in their activities on numerous alcohols, and since aging and heat-treatment experiments failed to reveal a stability difference, it is proposed that the difference is regulatory in nature, affecting ADH synthesis and primarily cis-acting. A survey of wild flies revealed additional variation in the FF/SS activity ratio. Further genetic analysis showed that the basis of this variation is not restricted to the second chromosome. Furthermore, modification of the activity ratio implies some degree of allelespecificity on the part of the modifiers.This work was supported in part by money collected by Jewish Community of Iowa City, Iowa, and by NSF Grant 76-01903 to Roger Milkman.     

Extension of life duration by dietary ethanol in Drosophila melanogaster: response to selection in two strains of different origins

Lines homozygous for the Adh ^S and Adh ^F alleles were extracted from a French and an African natural population of D. melanogaster. The lines from each geographic region were then crossed and the Mendelian F₂ constituted the first generation subjected to selection for an increase in adult survival in the presence of 2% ethanol.The responses to selection were similar in the two strains, in spite of their different ethanol tolerance. In less than 10 generations, life span with ethanol increased from about 7 to more than 12 days, with realized heritabilities of 0.14 and 0.18. This extension of longevity was also observed with other concentrations of ethanol but not with water alone. The increase in life span in presence of alcohol appears to result from unknown metabolic processes, which are not obligatorily related to the capacity of the flies to tolerate starvation, nor to their size, their lipid content of their ethanol tolerance. In the two lines, however, the Adh ^S allele was quickly eliminated, suggesting a selective advantage for the F allele.     

Synchronous allelic expression at the glucosephosphate isomerase A and B loci in interspecific sunfish hybrids

Allelic isozymes of glucosephosphate isomerase at the Gpi-A and -B loci were separated by starch gel electrophoresis in the warmouth (Lepomis gulosus) and green sunfish (L. cyanellus). The specific tissue distributions and developmental expressions of the GPI-A₂, -AB, and -B₂ isozymes were not different between these two species. The synchrony of allelic expression in normal intraspecific sunfish crosses was demonstrated by means of an electrophoretic variant at the Gpi-B locus. In embryos formed from warmouth × green sunfish hybrid crosses, the paternal GPI-A₂ isozymes were first expressed at the same time in both reciprocal hybrids, at 21–25 hr after fertilization. The maternal and paternal GPI-B subunits were synchronously expressed in reciprocal hybrids just prior to hatching. The parental allelic isozymes at both loci showed codominant expression in all tissues of the mature F₁ hybrids. These results are consistent with the absence of allelic asynchrony and inhibition in interspecific hybrids formed from more evolutionarily related species.     

Simultaneous induction by anaerobiosis or 2,4-D of multiple enzymes specificed by two unlinked genes: differential Adh1-Adh2 expression in maize

Summary The data of this paper partially define a two-gene, differentially inducible enzyme system in maize. Four major conclusions are drawn. (1) Anaerobic treatment results in the simultaneous expression of two unlinked Adh genes, Adh1 and Adh2. Adh2 is scarcely expressed in the uninduced root and negligibly expressed in the quiescent embryo. (2) The zero-order rate of anaerobic ADH protein induction reflects the zero-order rate of ADH synthesis throughout the induction; all active ADH isozymes display negligible turnover during the induction. (3) The auxin, 2,4-D, overcomes repression by air. The aerobic induction rates are also zero-order with negligible turnover for any isozyme. (4) The ratio of Adh1-Adh2 expression is drastically altered dependent on the mode of induction. The level of this differential regulatory phenomenon is before or at the assembly of ADH polypeptides into enzymologically or antigenically active dimers.     

The inheritance of tetraploid wheat seed peroxidases

Summary Embryo and endosperm peroxidases from dry mature seeds of three subspecies of tetraploid wheat (Triticum turgidum L.) were subjected to genetic analysis. The inheritance of eight isozymes (embryo isozymes a₂, d₁, d₂, e and f; and endosperm isozymes b, d and 4) were studied in F₂'s obtained from different wheat accessions. Simple monogenic inheritance producing three banded: one null segregation and two epistatic segregations (97 and 151) were found. In the case of isozymes b, d and 4, monogenic or epistatic segregation depended on the F₂ analyzed. Segregation data indicated that at least 9 different loci would determine the peroxidase isozymes of tetraploid wheat seed, all the loci studied containing null alleles. Furthermore, several loci determining embryo peroxidases were noticed to be mutually linked. All these data are discussed in context of the inheritance of seed peroxidases in hexaploid wheat and rye.