An electrophoretically cryptic alcohol dehydrogenase variant in Drosophila melanogaster. I. Activity ratios, thermostability, genetic localization and comparison with two other thermostable variants

The alcohol dehydrogenase (ADH) variant ADH-FCh.D. has a secondary alcohol/primary alcohol activity ratio characteristic of ADH-S although it has an electrophoretic mobility inseparable from ADH-F. ADH-FCh.D. is distinguished from these two common ADH variants by being much more thermostable. Genetic analysis suggests tht ADH-FCh.D. is specified by an allele at the Adh locus. Biochemical comparisons show that ADH-FCh.D. has the same electrophoretic mobility, activity ratio and thermostability as the two other heat-resistant variants which have been reported, ADH-F71K in Europe and ADH-Fr in North America. The geographically widespread distribution of a thermostable ADH variant within the ADH-F electrophoretic class indicates that it should be considered in attempts to explain the Adh polymorphism in natural populations.     

Locus Adh and adaptation of cn and vg mutants in the experimental populations of Drosophila melanogaster Meig

Complex study of adaptation and allozyme belonging of alcoholdehydrogenase (ADH) in cn and vg mutants has been carried out in the initial pure lines, in their panmixia populations and in condition of substitution of the mutant genotype by saturating crossings. It was shown that the high level of adaptation of cn mutants and the low level of adaptation of vg mutants was combined with the presence of different ADH allozymes. During the saturating crossings the reliable coadaptation of the genes cn and Adh(S) as well as vg and Adh(F) was detected that confirmes the postulated earlier conception of gene adaptation complexes.     

Alcohol dehydrogenase and adaptation to ethanol in Drosophila

The subject of this research is activity and allozyme spectra of alcohol dehydrogenase (ADH), and survival of mutant strains of Drosophila kept in standard nutrient medium with added ethanol. In all experiments the ADH of flies revealed greater affinity to isopropanol than ethanol. The mutant strains considerably differed from one another and from the wild type of flies in the level of enzyme activity, which may be connected with genotypic properties in the mutants studied. The ADH variability in mutant strains seems to be caused by different alleles of the structural ADH gene, which was established as a result of investigation of activity, electrophoretic mobility and thermostability of corresponding allozymes. As follows from experiments on the genotypical structure of populations in the conditions of fly selection in the medium containing ethanol (10%), the adaptation of flies to exogenous ethanol takes place via mechanisms of allele control of the ADH activity. Phenotypical manifestation of the ADH locus and its effect on the resistance of Drosophila to alcohol are supposed to depend on complex gene interactions determined by the genotype as a whole.     

Variation between electrophoretically identical alleles at the alcohol dehydrogenase locus in Drosophila melanogaster

A new variant of alcohol dehydrogenase (ADH 7lk) was found in a laboratory stock of Drosophila melanogaster. ADH in this stock had the same electrophoretic mobility as the F variant both on acrylamide and on agar. Activity levels were similar to the levels in F flies at temperature between 15 and 25 C. But while ADH F enzyme is inactivated rapidly at 40 C, ADH 7lk is still active. Also, ADH S is not inactivated at this temperature, but has a far lower activity per fly than ADH 7lk. Genetic analysis showed that the new variant is an allele of the Adh locus.     

Molecular analysis of a somaclonal mutant of maize alcohol dehydrogenase

Summary Plants regenerated from tissue cultures of maize were screened for variants of ADH1 and ADH2. Root extracts of 645 primary regenerant plants were tested, and one stable mutant of Adh1 was detected. The mutant gene (Adh1-Usv) produces a functional enzyme with a slower electrophoretic mobility than that of the progenitor Adh1-S allele, and is stably transmitted to progeny. The mutant was not present among four other plants derived from the same immature embryo, and therefore arose as a consequence of the culture procedure. The gene of Adh1-Usv was cloned and sequenced. A single base change in exon 6 was the only alteration found in the gene sequence. This would translate in the polypeptide sequence to a valine residue substituting for a glutamic acid residue, resulting in the loss of a negative charge and the production of a protein with slower electrophoretic mobility.Abbreviations kb kilobase pairs - ADH alcohol dehydrogenase     

The gene-enzyme system of alcohol dehydrogenase during genotype changes in Drosophila melanogaster]

It is established, that certain variants of replacement of chromosomes performed on wild (C-S, D) and mutant (cn, vg) lines as well as continuous backcrosses, leads to the change of heat resistance and activity of F- and S-allozymes of ADH in tissues of synthesized forms of drosophila. But nevertheless, the electrophoretic mobility of allozymes does not change. It is assumed that post-translated modifications of ADH play the important part in processes of phylogenetic adaptation of Drosophila melanogaster.     

Excessive vitamin A toxicity in mice genetically deficient in either alcohol dehydrogenase Adh1 or Adh3.

Alcohol dehydrogenase (ADH) deficiency results in decreased retinol utilization, but it is unclear what physiological roles the several known ADHs play in retinoid signaling. Here, Adh1, Adh3, and Adh4 null mutant mice have been examined following acute and chronic vitamin A excess. Following an acute dose of retinol (50 mg.kg(-1)), metabolism of retinol to retinoic acid in liver was reduced 10-fold in Adh1 mutants and 3.8-fold in Adh3 mutants, but was not significantly reduced in Adh4 mutants. Acute retinol toxicity, assessed by determination of the LD(50) value, was greatly increased in Adh1 mutants and moderately increased in Adh3 mutants, but only a minor effect was observed in Adh4 mutants. When mice were propagated for one generation on a retinol-supplemented diet containing 10-fold higher vitamin A than normal, Adh3 and Adh4 mutants had essentially the same postnatal survival to adulthood as wild-type (92-95%), but only 36% of Adh1 mutants survived to adulthood with the remainder dying by postnatal day 3. Adh1 mutants surviving to adulthood on the retinol- supplemented diet had elevated serum retinol signifying a clearance defect and elevated aspartate aminotransferase indicative of increased liver damage. These findings indicate that ADH1 functions as the primary enzyme responsible for efficient oxidative clearance of excess retinol, thus providing protection and increased survival during vitamin A toxicity. ADH3 plays a secondary role. Our results also show that retinoic acid is not the toxic moiety during vitamin A excess, as Adh1 mutants have less retinoic acid production while experiencing increased toxicity.     

Variation in the biochemical properties of the Drosophila alcohol dehydrogenase allozymes

Thirteen Drosophila Adh variants have been characterized with respect to gene expression, substrate preference, thermostability, and specific activity. The results suggest that the variants may be grouped into two biochemical classes, typified by the properties of the two most common enzyme forms, ADH-F and ADH-S. Membership of these classes cannot be predicted from electrophoretic mobility, nor is any simple classification possible with regard to the characteristics of level of gene expression (in terms of ADH activity or ADH protein) or thermostability of the gene product.     

Hypoxic Induction of Anoxia Tolerance in Roots of Adh1 Null Zea mays L

Seedlings of alcohol dehydrogenase 1 null mutants (Adh1-) of Zea mays L., which fail to synthesize alcohol dehydrogenase 1 (ADH1) isozymes, were hypoxically acclimated by 18 h of exposure to an atmosphere of 4% (v/v) O2 in N2 at 25[deg]C. Their ability to tolerate subsequent anoxia by exposure to anaerobic (O2-free) conditions was compared with that of unacclimated seedlings that were transferred immediately from an atmosphere of 40% (v/v) O2 to anaerobic conditions. Only 10% of the root tips of unacclimated seminal roots survived 6 h of anoxia, whereas 70% of the hypoxically acclimated root tips were viable at 24 h. During anoxia, acclimated root tips had enhanced ADH activity compared with unacclimated root tips, through induction of Adh2. Despite this, enzyme activity was still only about 5% that of acclimated, wild-type root tips and about half that of unacclimated, wild-type root tips. During anoxia, acclimated Adh1- root tips showed a higher rate of anaerobic respiration and ethanol production, greater concentrations of ATP and total adenylates, and a greater adenylate energy charge compared with unacclimated root tips. These results suggest that although enhanced ADH activity may have raised fermentation rates in acclimated Adh1- tissues and thereby contributed to energy metabolism and viability, the high levels of ADH activity inducible in acclimated, wild-type maize root tips appear to be in excess of that required to increase rates of fermentation.     

Anaerobic tolerant null: a mutant that allows Adh1 nulls to survive anaerobic treatment

Anaerobic tolerant null (ATN) is a recessive factor that allows alcohol dehydrogenase-1 (ADH1) null individuals of Zea mays L. to survive 24 h of anaerobic conditions. ADH1 null lines that do not possess this factor survive only a few hours of anoxia. We studied ADH activity levels in protein extracts from the primary root tissue of ATN. ADH levels were similar in ATN and other ADH1 null lines, suggesting that ADH activity does not account for differences in the ability of ATN to survive anaerobic treatment. The ATN survival trait segregated as a single recessive locus in crosses between ATN and double null (Adh1-S5657, Adh2-33). We also made crosses between ATN and 1s2p, an inbred line with ADH1 activity that carries an electrophoretic mutation of Adh2, to determine whether atn increases the number of survivors over that which would be expected from the segregation of Adh1 alone and to use the Adh2P allele to study the cosegregation of Adh2 and atn. The observed number of survivors in that cross exceeded the expected number of survivors by a margin consistent with a single recessive gene adding to the ADH+ survivors. Extracts from the primary root or scutellum of induced F2 seedlings from the above crosses were assayed for ADH activity by native polyacrylamide gel electrophoresis (PAGE) and simultaneously scored for survival to determine whether Adh2 and atn were segregating independently. We screened the (ATN x 1s2p)F2 progeny for ADH1 activity by staining root tips with an ADH-specific stain to select Adh1 null individuals prior to gel assay. Atn was found to be assorting independently of Adh1 and Adh2 in both crosses.     

Distinct retinoid metabolic functions for alcohol dehydrogenase genes Adh1 and Adh4 in protection against vitamin A toxicity or deficiency revealed in double null mutant mice

The ability of class I alcohol dehydrogenase (ADH1) and class IV alcohol dehydrogenase (ADH4) to metabolize retinol to retinoic acid is supported by genetic studies in mice carrying Adh1 or Adh4 gene disruptions. To differentiate the physiological roles of ADH1 and ADH4 in retinoid metabolism we report here the generation of an Adh1/4 double null mutant mouse and its comparison to single null mutants. We demonstrate that loss of both ADH1 and ADH4 does not have additive effects, either for production of retinoic acid needed for development or for retinol turnover to minimize toxicity. During gestational vitamin A deficiency Adh4 and Adh1/4 mutants exhibit completely penetrant postnatal lethality by day 15 and day 24, respectively, while 60% of Adh1 mutants survive to adulthood similar to wild-type. Following administration of a 50-mg/kg dose of retinol to examine retinol turnover, Adh1 and Adh1/4 mutants exhibit similar 10-fold decreases in retinoic acid production, whereas Adh4 mutants have only a slight decrease. LD(50) studies indicate a large increase in acute retinol toxicity for Adh1 mutants, a small increase for Adh4 mutants, and an intermediate increase for Adh1/4 mutants. Chronic retinol supplementation during gestation resulted in 65% postnatal lethality in Adh1 mutants, whereas only approximately 5% for Adh1/4 and Adh4 mutants. These studies indicate that ADH1 provides considerable protection against vitamin A toxicity, whereas ADH4 promotes survival during vitamin A deficiency, thus demonstrating largely non-overlapping functions for these enzymes in retinoid metabolism.     

Mutant alleles for hypoxanthine phosphoriboxyltransferase: codominant expression, complementation, and segregation in hybrid Chinese hamster cells.

Chinese hamster ovary cell mutants resistant to the purine analogs 6-thioguanine or 8-azaguanine have been isolated following mutagenesis with ethyl methane sulfonate. The activities of hypoxanthine phosphoribosyltransferase (HPRT) in three such mutants have been found to exhibit an increased Km for the substrate 5-phosphoribosyl-1-pyrophosphate. The isoelectric point of the mutant enzyme activity has also changed in two mutants. Hybrid cells containing one mutant and one wild-type allele express both genes. Segregants that have lost only the wild-type allele can be selected on the basis of drug resistance. Two mutants exhibiting different alterations in HPRT activity can complement in a hybrid cell to yield a wild-type growth pattern and enzyme activity with intermediate electrophoretic and kinetic properties. The results suggest intracistronic complementation between structural gene mutants of HPRT.     

Identification of a genetic element that controls the organ-specific expression of adh1 in maize

Allozyme balances serve as markers of quantitative behavior of electrophoretically distinguishable alleles. By the use of ADH Set I allozyme balances, it is demonstrated that all Adh1-S/Adh1-F individuals from more than 20 diverse S/F families exhibit a reciprocal correlation between Adh1 quantitative behavior in two maize organs: the scutellum and primary root. Within an electrophoretic mobility class, the Adh1 allele that is relatively underexpressed in the scutellum is relatively overexpressed in the primary root, and vice versa. Segregation tests prove that this "reciprocal effect" is the property of a cis-acting site that is closely linked to or within the Adh1 structural gene, and it is not affected by diverse genetic backgrounds. Immunological and [(3)H]-leucine incorporation experiments establish that Adh1 quantitative variants differ in ADH1.ADH1 synthetic rates in the anaerobic primary root. The reciprocal-effect phenomenon suggests that the cis-acting loci controlling Adh1 quantitative expression in each respective organ are at least in close proximity, or may share common DNA sequences. We discuss the possibility that the reciprocal-effect locus is a regulatory component of the Adh1 cistron.     

Correct developmental expression of a cloned alcohol dehydrogenase gene transduced into the Drosophila germ line

We have used P-element-mediated transformation to introduce a cloned Drosophila alcohol dehydrogenase (Adh) gene into the germ line of ADH null flies. Six independent transformants expressing ADH were identified by their acquired resistance to ethanol. Each transformant carries a single copy of the cloned Adh gene in a different chromosomal location. Four of the six transformant lines exhibit normal Adh expression by the following criteria: quantitative levels of ADH enzyme activity in larvae and adults; qualitative tissue specificity; the size of stable Adh mRNA; and the characteristic developmental switch in utilization of two different Adh promoters. The remaining two transformants express ADH enzyme activity with the correct tissue specificity, but at a lower level than wild type. These results demonstrate that an 11.8 kb chromosomal fragment containing the Adh gene includes the cis-acting sequences necessary for its correct developmental expression, and that a variety of chromosomal sites permit proper Adh gene function.     

Effect of colchicine and Triton X-100 on expression of the enzyme-encoding genes in nongerminating seeds of sugarbeet (Beta vulgaris L.)

The expression of the enzyme-coding genes, controlling glucose-phosphate isomerase (GPI), malate dehydrogenase (MDH), and alcohol dehydrogenase (ADH), was examined in nongerminating seeds of sugarbeet after Triton X-100 (TX-100) and colchicine treatment. Two types of changes revealed included modification of the enzymatic loci expression (change of the isozyme electrophoretic mobility) and inactivation of standard profiles. In the MDH and GPI systems, these processes were found to be associated. Complete isozyme modification was accompanied with the disappearance of standard profiles. In the ADH system, the treatment with TX-100 and colchicine gave rise to two independent processes, including silencing of the Adh1 locus and the appearance of the ADH isozymes with abnormal electrophoretic mobility, which were probably the products of the Adh2 locus. It was suggested that the effect of TX-100 and colchicine on the expression of the enzyme-encoding genes examined depended on the intracellular localization of the encoded enzymes.     

Gene-enzyme system of alcohol dehydrogenase and adaptation of Drosophila to increased temperature]

Properties and allelic ADH (alcohol dehydrogenase) control in Drosophila melanogaster were studied upon flies' cultivation under conditions of hypotherm of different intensity and duration. Lines homozygotic for F allele (vg) and S allele (cn) of the Adh gene as well as genetically enriched experimental cn' and vg' populations containing a small number of AdhF/AdhS heterozygotes at the initial stage were used. It was found out that physiological adaptation of the species to momentary influence of elevated temperature is accompanied by modification of physical properties of ADH-F according to ADH-S. Constant influence (during the life span of 25 generations) of elevated temperature on the population changes its genetic structure, due to selective advantages of the S allele of Adh under these conditions.     

Intragenic recombination in maize: pollen analysis methods and the effect of parental adh1 isoalleles

The ability to stain mature pollen grains for the presence of alcohol dehydrogenase (ADH) activity permits the quantitation of ADH( +) gametophytes at frequencies below 10(-6). This resolution allows reversion and genetic fine structure analyses. The rationale of pollen analysis follows Nelson's prototype studies with waxy. As with the waxy gene, revertant frequencies for seven Adh1-deficient ( Adh1(-)) alleles appear to be in excess of microbially derived expectations. Each of the seven Adh1(-) alleles were derived from one of three naturally occurring isoalleles. Based on Schwartz's protein level characterizations of the mutants' products, it was anticipated that the seven Adh1(-) alleles should recombine to yield ADH(+) cistrons in certain pairwise combinations. This expectation was not met. The parental "wild-type" isoalleles from which the mutants were derived appear to be structurally divergent. The discussion interprets these data in view of understanding naturally occurring cistronic variation.     

Developmental profile and tissue distribution of Drosophila alcohol dehydrogenase: an immunochemical analysis with monoclonal antibodies.

To analyze Drosophila alcohol dehydrogenase gene (Adh) expression and tissue distribution at various developmental stages, we devised several immunochemical techniques making use of monoclonal antibodies against Drosophila alcohol dehydrogenase (ADH), which had been obtained previously. We here report their application to analyze the expression of Adh in a wild-type strain of D. melanogaster. s-ELISA tests were performed to evaluate fluctuations in ADH content and specific activity during development in individual organs as well as in whole individuals. In all cases, ADH specific activity appeared to be quite constant, which implies that variations in enzyme activity reflect differences in protein content. Immunoblottings of crude homogenates revealed immunoreactive low relative molecular mass peptides in addition to the 27 KD monomeric band, showing a conserved banding pattern in different organs and developmental stages. Immunohistochemical assays on whole organs were used to analyze the general pattern of ADH distribution. Immunoperoxidase staining of cryosections proved to be of crucial relevance, as it yielded full details of the tissue localization of ADH within the ADH-positive organs. We have shown not only that ADH displays a specific distribution in some organs but also that the enzyme is restricted to certain cell types.     

Expression of the enzyme-encoding genes under the influence of colchicine and triton X-100 in nongerminating seeds of sugarbeet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.)

The expression of the enzyme-coding genes, controlling glucose-phosphate isomerase (GPI), malate dehydrogenase (MDH), and alcohol dehydrogenase (ADH), was examined in nongerminating seeds of sugarbeet after Triton X-100 (TX-100) and colchicine treatment. Two types of changes revealed included modification of the enzymatic loci expression (change of the isozyme electrophoretic mobility) and inactivation of standard profiles. In the MDH and GPI systems, these processes were found to be associated. Complete isozyme modification was accompanied with the disappearance of standard profiles. In the ADH system, the treatment with TX-100 and colchicine gave rise to two independent processes, including silencing of the Adh1 locus and the appearance of the ADH isozymes with abnormal electrophoretic mobility, which were probably the products of the Adh2 locus. It was suggested that the effect of TX-100 and colchicine on the expression of the enzyme-encoding genes examined depended on the intracellular localization of the encoded enzymes.