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.     

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.     

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.     

The purification and biochemical properties of alcohol dehydrogenase—“Fast (chateau douglas)” from Drosophila melanogaster

Alcohol dehydrogenase has been purified from Drosophila melanogaster lines bearing the Adh ^F, Adh^S, and Adh ^FCh.D. alleles. Biochemical investigations show that the properties of the purified enzymes are very similar to those of crude enzyme extracts except that the pure enzymes are more heat stable. ADH-FCh.D. resembles ADH-S very closely in specific activity, substrate specificity, and a number of kinetic parameters including limiting values for K _m(app.) for ethanol. However, it is considerably more heat stable than either of the two common variants. ADH-F differs from ADH-S and ADH-FCh.D. particularly with regard to the rate of oxidation of secondary alcohols. Atomic absorbtion spectroscopy shows that all three allozymes lack zine or other divalent cations as active-site components. Peptide mapping experiments identify one very active cysteinyl residue; and amide residues in the NAD⁺ binding domain.     

Genetic regulation of liver alcohol dehydrogenase in Peromyscus

Data from genetic crosses of Peromyscus maniculatus and P. polionotus suggest that electrophoretic variants of liver alcohol dehydrogenase are coded by alleles at a single locus. These alleles, designated Adh ^F , Adh ^S , and Adh ^N , determine, respectively, the fast, slow, and not detectable (null) ADH electrophoretic phenotypes. Heterozygotes (Adh ^F /Adh ^S ) exhibit three bands on zymograms, suggesting a dimeric subunit structure for the enzyme. However, Adh ^F /Adh ^N and Adh ^S /Adh ^N animals exhibit a single band, suggesting that the Adh ^N allele does not produce a polypeptide subunit capable of dimerizing into an active molecule. Fast and slow electrophoretic phenotypes exhibit multiple bands which can be converted into single major fast and slow bands, respectively, upon treatment with oxidized or reduced NAD. Addition of NAD also stabilizes both the fast and slow enzyme to heat inactivation at 60 C for at least 30 min.This work was supported by Predoctoral Fellowship AA-05067 from the National Institute of Alcohol Abuse and Alcoholism to K. G. B. and South Carolina Commission on Alcohol and Drug Abuse Grant 7607. Also, partial support was provided by NIH Grant CA-16184.     

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.     

Selection against homozygotes in the ADH polymorphic system of Drosophila melanogaster associated with the lethal factor l(2) Stm

In the natural populations ⁺Tüb, ⁺Prov, and ⁺Rov, similar Adh ^F allele frequencies occur (q _F=0.11, 0.18, and 0.08, respectively). However, there is a discrepancy in that the Adh ^F allele in ⁺Tüb is closely linked to the lethal factor 1(2)Stm, which reduces relative fitness of the F phenotype to zero. In spite of this, polymorphism is maintained also in ⁺Tüb, because the heterozygotes are superior to the homozygous S type (relative fitness=0.88). Under laboratory culture conditions, in ⁺Tüb the relative fitness of the S genotype further decreases to 0.6. After outcrossing the lethal factor, relative fitnesses for S, FS, and F become 0.6, 1, and 0.48, respectively, implying that fitness for S remains the same. Relative values for S, FS, and F in ⁺Prov, not affected by the lethal factor, are calculated by the maximum average fitness method to be 1, 1.2, and 0.2 under the assumption that heterozygous FS are similarly superior to S as in the natural ⁺Tüb population and all allele frequencies found are stable equilibrium values.     

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.     

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.     

Adh n5: A temperature-sensitive mutant at the Adh locus in Drosophila

Individuals of an alcohol dehydrogenase-negative strain of Drosophila melanogaster designated Adh ⁿ⁵ are resistant to ethanol poisoning at low but not at high temperatures. The basis for this behavior is that Adh ⁿ⁵ flies contain a mutant form of alcohol dehydrogenase which is less heat stable than that of wild-type flies. The mutation in Adh ⁿ⁵ maps at, or very close to, the presumptive structural locus and is not complemented by any of 11 other alcohol dehydrogenase-null mutants.This research was supported by Grant No. GM 18254 from the National Institutes of Health and Grant No. M55.2217 from the National Cancer Institute.Publication No. 768 from the Department of Biology, Johns Hopkins University.     

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.     

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 II. Adh genotype frequency variations in Drosophila cage populations reared at different temperatures

The adaptive response of the Adh locus to different temperatures was determined in an experimental population with a stabilized gene pool in order to confirm the conclusions reached in a previous paper (Pieragostini et al., Genetica 56: 27–37, 1981) using as experimental material a laboratory population in which heterosis and strong interaction between loci were present.By sampling a stable lab population, three cage populations were constructed, heterozygous at the Adh locus and genetically identical but reared at different temperatures (P 18 °C, P 25 °C and P 28 °C). On these populations we studied the dynamics of Adh allele frequencies and of five metric traits up to the fifth generation of caging. The most interesting result is represented by the differences between P 28 °C and the other two populations (P 25 °C and P 18 °C). In particular P 25 °C and P 18 °C show a strong similarity with the original population both in Adh allele frequency and relationship between Adh genotype and the respective metric phenotype; this is not the case for P28 °C which differed from the original population and the other two newly founded ones not only in Adh allele frequencies but even in gene arrangements.Comparing these results with those obtained in the previous work it can be concluded that Adh frequency variations can be a side effect of the evolutionary pattern of the populations which in turn depends on the initial genetic composition.     

The alcohol dehydrogenase alleloenzymes AdhS and AdhF from the fruitfly Drosophila melanogaster: An enzymatic rate assay to determine the active-site concentration

A rapid and reproducible enzymatic rate assay for the quantitative determination of the concentration of active sites is presented for the alleloenzymes Adh^S and Adh^F from Drosophila melanogaster. Using this procedure the turnover numbers as catalytic-center activities were found to be 12.2 sec^–1 for Adh^F and 3.4 sec^–1 for Adh^S with secondary alcohols. This showed a slower dissociation of the coenzyme from the binary enzyme-NADH complex with Adh^S and hence a stronger binding of NADH to this alleloenzyme. With ethanol, the catalytic-center activity was 1.4 sec^–1 for Adh^S and 2.8 sec^–1 for Adh^F, and hence the single amino acid mutation distinguishing the two alleloenzymes also affected hydride transfer.     

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.     

Purification and characterization of an endoxylanase from the culture broth of <Emphasis Type="Italic">Bacillus cereus</Emphasis> BSA1

An extracellular xylanase from the fermented broth of Bacillus cereus BSA1 was purified and characterized. The enzyme was purified to 3.43 fold through ammonium sulphate precipitation, DEAE cellulose chromatography and followed by gel filtration through Sephadex-G-100 column. The molecular mass of the purified xylanse was about 33 kDa. The enzyme was an endoxylanase as it initially degraded xylan to xylooligomers. The purified enzyme showed optimum activity at 55°C and at pH 7.0 and remained reasonably stable in a wide range of pH (5.0–8.0) and temperature (40–65°C). The K _m and V _max values were found to be 8.2 mg/ml and 181.8 μmol/(min mg), respectively. The enzyme had no apparent requirement of cofactors, and its activity was strongly inhibited by Cu²⁺, Hg²⁺. It was also a salt tolerant enzyme and stable upto 2.5 M of NaCl and retained its 85% activity at 3.0 M. For stability and substrate binding, the enzyme needed hydrophobic interaction that revealed when most surfactants inhibited xylanase activity. Since the enzyme was active over wide range of pH, temperature and remained active in higher salt concentration, it could find potential uses in biobleaching process in paper industries.     

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.     

Cloning and sequence analysis of genes involved in erythromycin biosynthesis in Saccharopolyspora erythraea: sequence similarities between EryG and a family of S-adenosylmethionine-dependent methyltransferases.

Summary Treatment of tomato seeds with ethyl methanesulphonate (EMS) followed by allyl alcohol selection of M₂ seeds has led to the identification of one plant (B15-1) heterozygous for an alcohol dehydrogenase (Adh) null mutation. Genetic analysis and expression studies indicated that the mutation corresponded to the structural gene of the Adh-1 locus on chromosome 4. Homozygous Adh-1 null mutants lacked ADH-1 activity in both pollen and seeds. Using an antiserum directed against ADH from Arabidopsis thaliana, which crossreacts with ADH-1 and ADH-2 proteins from tomato, no ADH-1 protein was detected in seeds of the null mutant. Northern blot analysis showed that Adh-1 mRNA was synthesized at wild-type levels in immature seeds of the null mutant, but dropped to 25% in mature seeds. Expression of the Adh-2 gene on chromosome 6 was unaffected. The potential use of the Adh-1 null mutant in selecting rare transposon insertion mutations in a cross with mutable Adh-1 ⁺ tomato lines is discussed.     

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.     

The fate of several migrant genes in isolated populations of Drosophila melanogaster

Sepia-eyed flies carrying the slow electrophoretic variant of either Est-6 or Adh were introduced in low numbers and at infrequent intervals into populations of wildtype flies (+ ^se /+ ^se ) that were also homozygous for the fast moving variant of either Est-6 (50 populations) or Adh (50 populations). After 24 generations, the frequency of the sepia alleles was approximately 25%, although there was considerable variation from population to population. The fate of the Est-6 slow allele corresponded closely to that of sepia (which is located ten map units distant), although one population retained the slow allozyme variant but rejected sepia. The Adh slow allele was also retained by many populations. A number of them retained Adh-S but not sepia, and vice versa; these loci are on different chromosomes. The advantage of sepia heterozygotes was estimated to be about twice that of wildtype homozygotes. The data suggest that the selective advantage resides not with the sepia locus itself, but with a nearby chromosomal region.Financial support for work reported here was supplied under grant number GM24850, National Institutes of Health.