Adh-negative mutants: Detection of an altered tryptic peptide in a mutant enzyme of Drosophila

Adh^nll is an ethyl methanesulfonate induced mutant that lacks detectable alcohol dehydrogenase activity. A number of indirect lines of evidence have indicated that the mutation responsible for this loss in enzyme activity is locoalized in the Adh structural gene. We present more direct evidence for this hypothesis here. Utilizing a newly developed procedure for comparing tryptic peptides of Drosophila alcohol dehydrogenase obtained from different strains, we show that the alcohol dehydrogenase-like protein isolated from Adh ^nll exhibits an altered peptide profile when compared to that of wild type. The implications of this finding as well as the utility of the method for attacking other genetic and developmental problems are discussed.This work was supported by grants from the NIH (GM-18254 and ES-01527) and by a contract from the Department of Energy (EY-76-S-02-2965).Contribution No. 1050 from the Department of Biology, John Hopkins University, Baltimore, Maryland 21218.     

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.     

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.     

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.     

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.     

Genetic control of alcohol dehydrogenase levels in Drosophila

Among the progeny of Drosophila flies heterozygous for two noncomplementing Adh-negative alleles, two individuals were found that had recovered appreciable alcohol dehydrogenase activity, thereby surviving the ethanol medium used as a screen. The most likely explanation is that these Adh-positive flies are the product of intracistronic recombination within the Adh locus. Judging by the distribution of outside markers, one of the crossovers would have been a conventional reciprocal exchange while the other appears to have been an instance of nonreciprocal recombination. The enzymes produced in strains derived from the original survivors can be easily distinguished from wild-type enzymes ADH-S and ADH-F on the basis of their sensitivity to denaturing agents. None of various physical and catalytic properties tested revealed differences between the enzymes of the survivor strains except that in one of them the level of activity is 55–65% of the other. Quantitative immunological determinations of ADH gave estimates of enzyme protein which are proportional to the measured activity levels. These results are interpreted to indicate that different amounts of ADH protein are being accumulated in the two strains.This work was supported in part by NSF Grant PCM 76-19563.     

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.     

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.     

Cytosolic calcium is involved in the regulation of barley hemoglobin gene expression

Hemoglobin gene expression is upregulated during hypoxia. To determine whether the induction occurs via similar mechanisms that have been proposed for other hypoxically induced proteins, barley (Hordeum vulgare L.) aleurone layers were treated with various agents that interfere with known components of signal transduction. Ruthenium red, an organelle calcium channel blocker, inhibited anoxia-induced hemoglobin (Hb) and alcohol dehydrogenase (EC 1.1.1.1) (Adh) gene expression in a dose-dependent manner. The divalent ionophore, A23187, combined with EGTA also dramatically reduced anoxia-induced Hb and Adh expression. Normal induction of Hb by anoxia in EGTA-treated cells was restored by adding exogenous Ca²⁺ but not Mg²⁺, suggesting that cytosolic calcium is involved in Hb and Adh regulation. W-7, a calmodulin antagonist, did not affect anaerobically induced Hb and Adh expression even though it induced Hb under aerobiosis. A3, a protein kinase inhibitor, did not significantly affect anaerobically induced Hb, but did significantly upregulate the gene under aerobic conditions. The results indicate that calmodulin-independent anaerobic alteration in cytosolic Ca²⁺ and protein dephosphorylation are factors in Hb induction.     

Variation in alcohol dehydrogenase activity in vitro in flies from natural populations ofDrosophila melanogaster

Alcohol dehydrogenase (ADH) activity variation in male flies taken directly from seven natural populations ofDrosophila melanogaster is largely accounted for by segregation of alleles at theAdh structural gene locus. There was little overlap in the ADH activities ofAdh ^F andAdh ^s homozygotes. Body weights varied only slightly betweenAdh genotypes and contributed little to ADH variation. Between and within population variation in ADH activity and ADH protein in flies in the wild is mainly due to the relative frequencies ofAdh ^F andAdh ^s.     

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.     

Isozyme variability in species of the genus Drosophila. VI. Frequency-property-environment relationships of allelic alcohol dehydrogenases in D. melanogaster

Adult Drosophila melanogaster from naturally occurring populations in the Eastern United States were examined by gel electrophoresis for their alcohol dehydrogenase (ADH) phenotype. The ADH enzymes were partially purified and characterized. Frequencies of the controlling alleles, Adh ⁴ and Adh ⁶, were discovered to vary in a clinal pattern. Adh ⁶ reaches a maximum frequency of about 0.90 in the South and minimum of about 0.50 in the North. Partially purified enzymes from the three Adh genotypes varied according to specific activity, substrate specificity, and heat stability. A differential influence of pH was indicated. There was little variation in K _m values for ethanol and DPN⁺ among the enzymes.This work was supported by AEC Contract No. AT-(40-1)-3980 and by PHS Research Grant No. GM 11546.Paper No. 3880 of the Journal Series of the North Carolina Experiment Station, Raleigh, North Carolina. This work incorporates, in part, the thesis research of C. L. Vigue to be submitted in partial fulfillment of the Ph.D. requirements in Genetics.     

Adaptive significance of the action of the Drosophila melanogaster alcohol dehydrogenase locus through the heat shock protein system

Four Drosophila melanogaster strains, each homozygous for one of the two major ADH aliozymes, Fast and Slow (Adh ^F1, Adh ^S , Adh ^F2 and Adh ^S2) were used to study the interaction of the Adh locus with ethanol and temperature. The separate and especially the combined effects of these two parameters allow the conclusion that the Adh locus of D. melanogaster intervenes in the adaptation process through the heat shock protein system.     

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.     

Perturbation of gene frequencies in a natural population of Drosophila melanogaster: evidence for selection at the Adh locus

The cellar population of Drosophila melanogaster at the Chateau Tahbilk Winery (Victoria, Australia) was perturbed for alcohol dehydrogenase (Adh) gene frequencies. Phenol oxidase (Phox) frequencies were also perturbed and monitored as a control. Subsequent gene frequency changes, together with information on population structure, indicated that selection acted on the chromosome regions of both loci. Adh gene frequencies returned to preperturbation levels in a predictable manner. A model in which the relative fitness of Adh phenotypes was determined by temperature-dependent specific activities of enzymes of Adh genotypes adequately accounts for the rate of gene frequency change at this locus. Thus temperature behaves as a selective agent in modulating Adh gene frequencies in this cellar environment.     

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.     

Alcohol dehydrogenase polymorphism in populations of Drosophila melanogaster. II. Relation between ADH activity and adult mortality

Eight Drosophila melanogaster strains, seven homozygous for Adh ^F alleles and one for an Adh-null mutant, were compared for ADH activity in males and adult mortality on ethanol-supplemented food. The strains differed considerably in these qualities. A positive correlation was found between ADH activity and ld ₅₀. The relevance of this finding is discussed in relation to the differential selection acting on Adh genotypes kept on ethanol-supplemented food.     

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.     

Alcohol dehydrogenase polymorphism in the seaweed fly,Coelopa frigida

Seaweed flies (Coelopa frigida) inhabit piles of decaying seaweed on the seashore. All populations so far studied have been found to be polymorphic at the alcohol dehydrogenase locus (Adh). This article reports an attempt to identify some of the forces of natural selection that may be maintaining this polymorphism. First, the genetic determination of the rather complex isozyme system is described. Several inbred lines homozygous at the Adh locus were derived and the biochemical properties of their allozymes compared. Significant differences in both specific activities and thermal stabilities were found between ADH allozymes. A simple experiment is reported in which individuals with different Adh genotypes were cultured in competition with each other in the presence of elevated levels of ethanol. Although the presence of ethanol resulted in greater mortality, there is no evidence that it was selective with respect to the Adh genotypes. The possible relevance of these results to the maintenance of the Adh polymorphism is discussed.This work was supported by a grant to T. H. D. from the Science Research Council.