ADH Protein Research Articles

Proteomic Studies of Roots in Hypoxia-Sensitive and -Tolerant Tomato Accessions Reveal Candidate Proteins Associated with Stress Priming.

Tomato (Solanum lycopersicum L.) is a vegetable frequently exposed to hypoxia stress induced either by being submerged, flooded or provided with limited oxygen in hydroponic cultivation systems. The purpose of the study was to establish the metabolic mechanisms responsible for overcoming hypoxia in two tomato accessions with different tolerance to this stress, selected based on morphological and physiological parameters. For this purpose, 3-week-old plants (plants at the juvenile stage) of waterlogging-tolerant (WL-T), i.e., POL 7/15, and waterlogging-sensitive (WL-S), i.e., PZ 215, accessions were exposed to hypoxia stress (waterlogging) for 7 days, then the plants were allowed to recover for 14 days, after which another 7 days of hypoxia treatment was applied. Root samples were collected at the end of each time-point and 2D-DIGE with MALDI TOF/TOF, and expression analyses of gene and protein-encoded alcohol dehydrogenase (ADH2) and immunolabelling of ADH were conducted. After collating the obtained results, the different responses to hypoxia stress in the selected tomato accessions were observed. Both the WL-S and WL-T tomato accessions revealed a high amount of ADH2, which indicates an intensive alcohol fermentation pathway during the first exposure to hypoxia. In comparison to the tolerant one, the expression of the adh2 gene was about two times higher for the sensitive tomato. Immunohistochemical analysis confirmed the presence of ADH in the parenchyma cells of the cortex and vascular tissue. During the second hypoxia stress, the sensitive accession showed a decreased accumulation of ADH protein and similar expression of the adh2 gene in comparison to the tolerant accession. Additionally, the proteome showed a greater protein abundance of glyceraldehyde-3-phosphate dehydrogenase in primed WL-S tomato. This could suggest that the sensitive tomato overcomes the oxygen limitation and adapts by reducing alcohol fermentation, which is toxic to plants because of the production of ethanol, and by enhancing glycolysis. Proteins detected in abundance in the sensitive accession are proposed as crucial factors for hypoxia stress priming and their function in hypoxia tolerance is discussed.

Analysis of alcohol dehydrogenase activity and fermentation characteristics of Acetobacter pasteurianus JST-S strain

Alcohol dehydrogenase activity (ADH), acetic acid yield, and tolerance to temperature and acid-induced stress are important bacterial strain parameters for the industrial production of acetic acid or vinegar. In this study, we evaluated and compared multiple features between A. pasteurianus JST-S strain, screened in the laboratory, and A. pasteurianus CICC 20001, a commonly used industrial strain. The ADH enzyme activity peaked at 8.22 U mg−1 for JST-S compared with the 7.62 U mg−1 for CICC 20001. Further, the ADH protein level was higher in JST-S than in the CICC 20001 strain. Comparative analysis of growth and cell morphology of the two strains indicated that the acetic acid tolerance of JST-S is superior to that of CICC 20001. Further, when the two strains were used for semi-continuous fermentation in 4 batches, the total acid production in fermentation broth with the JST-S peaked at 62.96 ± 1.42 g L−1 compared with 56.83 ± 1.12 g L−1 for the CICC 20001 strain. Thus, the JST-S strain seems to have better fermentation characteristics than the commonly used industrial strain. Based on all our observations, we propose that A. pasteurianus JST-S may be applied for cost-effective industrial production to obtain a high concentration of acetic acid.

Stress-Induced Detoxification Enzymes in Rice Have Broad Substrate Affinity.

Reactive carbonyl compounds (RCCs) such as hydroxynonenol, malondialdehyde, acrolein, crotonaldehyde, methylglyoxal, and glyoxal accumulate at higher levels under stress in plants and damage the cell metabolic activities. Plants have evolved several detoxifying enzymes such as aldo–keto reductases (AKRs), aldehyde/alcohol dehydrogenases (ALDH/ADH), and glyoxalases. We report the phylogenetic relationship of these proteins and in silico analysis of rice-detoxifying protein structures and their substrate affinity with cofactors using docking and molecular simulation studies. Molecular simulations with nicotinamide adenine dinucleotide phosphate or glutathione cofactor docking with commonly known reactive substrates suggests that the AKRs, ALDH, and ADH proteins attain maximum conformational changes, whereas glyoxalase has fewer conformational changes with cofactor binding. Several AKRs showed a significant binding affinity with many RCCs. The rice microarray studies showed enhanced expression of many AKRs in resistant genotypes, which also showed higher affinity to RCCs, signifying their importance in managing carbonyl stress. The higher expression of AKRs is regulated by stress-responsive transcription factors (TFs) as we identified stress-specific cis-elements in their promoters. The study reports the stress-responsive nature of AKRs, their regulatory TFs, and their best RCC targets, which may be used for crop improvement programs.

The monoclonal antibody Ca37, developed against Candida albicans alcohol dehydrogenase, inhibits the yeast in vitro and in vivo

Candida albicans is a commensal yeast able to cause life threatening invasive infections particularly in immunocompromised patients. Despite the availability of antifungal treatments, mortality rates are still unacceptably high and drug resistance is increasing. We, therefore, generated the Ca37 monoclonal antibody against the C. albicans alcohol dehydrogenase (Adh) 1. Our data showed that Ca37 was able to detect C. albicans cells, and it bound to Adh1 in yeast and Adh2 in hyphae among the cell wall-associated proteins. Moreover, Ca37 was able to inhibit candidal growth following 18 h incubation time and reduced the minimal inhibitory concentration of amphotericin B or fluconazole when used in combination with those antifungals. In addition, the antibody prolonged the survival of C. albicans infected-Galleria mellonella larvae, when C. albicans was exposed to antibody prior to inoculating G. mellonella or by direct application as a therapeutic agent on infected larvae. In conclusion, the Ca37 monoclonal antibody proved to be effective against C. albicans, both in vitro and in vivo, and to act together with antifungal drugs, suggesting Adh proteins could be interesting therapeutic targets against this pathogen.

A single Asp isomer substitution in an αA-crystallin-derived peptide induces a large change in peptide properties

The eye lens is mainly composed of crystallins, which undergo modifications such as oxidation, deamidation and isomerization with aging. Asp58, Asp76, Asp84, and Asp151 residues of αA-crystallin are site-specifically isomerized to L-iso, D-, and D-iso isomers in aged-related cataract lenses. In addition, an αA66–80 peptide, corresponding to the 66–80 (66SDRDKFVIFLDVKHF80) fragment of human αA-crystallin, is detected in aged lens. This peptide induces protein aggregation and causes loss of the chaperone function of α-crystallin. The αA66–80 peptide contains Asp76, but it is not known whether isomerization of Asp76 in αA66–80 specifically induces protein aggregation or affects α-crystallin function. Using Fmoc-based solid-phase synthesis, here we synthesized four αA66–80 peptides, each containing L-, L-iso, D-, or D-isoAsp at position 76, and compared their structures and properties. Normal αA66–80 peptide containing the L-Asp76 isomer increased the EDTA-induced aggregation of ADH protein, DTT-induced aggregation of insulin, and heat-induced aggregation of βL-crystallin. αA66–80 peptide containing D- or D-isoAsp76 had similar or no effects on the aggregation of these proteins. By contrast, αA66–80 peptide containing L-isoAsp76 inhibited the aggregation of all three proteins, indicating that it has chaperone activity. With regard to secondary structure, αA66–80 peptide containing the L-, D-, or D-isoAsp76 isomer had random-coil structure, whereas αA66–80 peptide containing L-isoAsp76 had β-sheet like structure. A Thioflavin T (ThT) assay indicated that only the L-isoAsp-containing αA66–80 peptide has β-sheet structure and generates amyloid fibrils. Collectively, these observations indicate that isomerization of Aps76 to the Lβ isomer endows β-sheet structure and chaperone function on this peptide.

Penicisulfuranol A, a novel C-terminal inhibitor disrupting molecular chaperone function of Hsp90 independent of ATP binding domain

The goal of this study is to explore the mechanism of a heat shock protein 90 (Hsp90) C-terminal inhibitor, Penicisulfuranol A (PEN-A), for cancer therapy. PEN-A was produced by a mangrove endophytic fungus Penicillium janthinellum and had a new structure with a rare 3H-spiro [benzofuran-2, 2′-piperazine] ring system. PEN-A caused depletion of multiple Hsp90 client proteins without induction of heat shock protein 70 (Hsp70). Subsequently, it induced apoptosis and inhibited xerograph tumor growth of HCT116 cells in vitro and in vivo. Mechanism studies showed that PEN-A was bound to C-terminus of Hsp90 at the binding site different from ATP binding domain. Therefore, it inhibited dimerization of Hsp90 C-terminus, depolymerization of ADH protein by C-terminus of Hsp90, and interaction of co-chaperones with Hsp90. These inhibitory effects of PEN-A were similar to those of novobiocin, an inhibitor binding to interaction site for ATP of C-terminus of Hsp90. Furthermore, our study revealed that disulfide bond was essential moiety for inhibition activity of PEN-A on Hsp90. This suggested that PEN-A may be bound to cysteine residues near amino acid region which was responsible for dimerization of Hsp90. All results indicate that PEN-A is a novel C-terminal inhibitor of Hsp90 and worthy for further study in the future not only for drug development but also for unraveling the bioactivities of Hsp90.

Computational studies of human class V alcohol dehydrogenase - the odd sibling.

BackgroundAll known attempts to isolate and characterize mammalian class V alcohol dehydrogenase (class V ADH), a member of the large ADH protein family, at the protein level have failed. This indicates that the class V ADH protein is not stable in a non-cellular environment, which is in contrast to all other human ADH enzymes. In this report we present evidence, supported with results from computational analyses performed in combination with earlier in vitro studies, why this ADH behaves in an atypical way.ResultsUsing a combination of structural calculations and sequence analyses, we were able to identify local structural differences between human class V ADH and other human ADHs, including an elongated β-strands and a labile α-helix at the subunit interface region of each chain that probably disturb it. Several amino acid residues are strictly conserved in class I–IV, but altered in class V ADH. This includes a for class V ADH unique and conserved Lys51, a position directly involved in the catalytic mechanism in other ADHs, and nine other class V ADH-specific residues.ConclusionsIn this study we show that there are pronounced structural changes in class V ADH as compared to other ADH enzymes. Furthermore, there is an evolutionary pressure among the mammalian class V ADHs, which for most proteins indicate that they fulfill a physiological function. We assume that class V ADH is expressed, but unable to form active dimers in a non-cellular environment, and is an atypical mammalian ADH. This is compatible with previous experimental characterization and present structural modelling. It can be considered the odd sibling of the ADH protein family and so far seems to be a pseudoenzyme with another hitherto unknown physiological function.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-016-0072-y) contains supplementary material, which is available to authorized users.

Adh enhances Actinobacillus pleuropneumoniae pathogenicity by binding to OR5M11 and activating p38 which induces apoptosis of PAMs and IL-8 release.

Members of the Trimeric Autotransporter Adhesin (TAA) family play a crucial role in the adhesion of Gram-negative pathogens to host cells, but the immunopathogenesis of TAAs remains unknown. Our previous studies demonstrated that Adh from Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is required for full bacterial pathogenicity. Alveolar macrophages are the first line of defense against respiratory infections. This study compared the interactions between porcine alveolar macrophages (PAMs) and wild-type A. pleuropneumoniae (5b WT) or an Adh-deletion strain (5b ΔAdh) via gene microarray, immunoprecipitation and other technologies. We found that Adh was shown to interact with the PAMs membrane protein OR5M11, an olfactory receptor, resulting in the high-level secretion of IL-8 by activation of p38 MAPK signaling pathway. Subsequently, PAMs apoptosis via the activation of the Fax and Bax signaling pathways was observed, followed by activation of caspases 8, 9, and 3. The immunological pathogenic roles of Adh were also confirmed in both murine and piglets infectious models in vivo. These results identify a novel immunological strategy for TAAs to boost the pathogenicity of A. pleuropneumoniae. Together, these datas reveal the high versatility of the Adh protein as a virulence factor and provide novel insight into the immunological pathogenic role of TAAs.

A case of primary adrenal diffuse large B cell lymphoma presenting with severe hyponatremia.

We herein report a case of primary adrenal lymphoma with severe hyponatremia. The patient was admitted for an evaluation of severe hyponatremia and an enlarged bilateral adrenal mass, which were found in a previous examination for causes of general fatigue and anorexia. Laboratory data, including the serum levels of sodium (115mEq/L), osmolality (239mOsm/kgH2O), ADH (5.8pg/mL), cortisol (11.6μg/dL), free T3 (2.42pg/mL), urinary Na (117mEq/L) and urine osmolality (490mOsm/kgH2O), fulfilled the diagnostic criteria for the syndrome of inappropriate secretion of ADH (SIADH). An abdominal computed tomography scan revealed a large bilateral adrenal mass. A biopsy of the enlarged left adrenal mass revealed diffuse large B cell lymphoma, which was negative for ADH protein. Hydrocortisone treatment normalized the patient's body temperature and serum sodium concentration. In this case, hyponatremia developed when both adrenal glands were involved and was normalized with hydrocortisone. These findings suggest that adrenal insufficiency was the cause of hyponatremia, although the basal serum cortisol was normal. The current case suggests that the administration of hydrocortisone is recommended if suspicious clinical signs or symptoms are found in severe hyponatremia, even if hyponatremia is associated with a normal serum cortisol level and fulfills the diagnostic criteria for SIADH.

Cofactor Specificity of the Bifunctional Alcohol and Aldehyde Dehydrogenase (AdhE) in Wild-Type and Mutant Clostridium thermocellum and Thermoanaerobacterium saccharolyticum.

Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are thermophilic bacteria that have been engineered to produce ethanol from the cellulose and hemicellulose fractions of biomass, respectively. Although engineered strains of T. saccharolyticum produce ethanol with a yield of 90% of the theoretical maximum, engineered strains of C. thermocellum produce ethanol at lower yields (∼50% of the theoretical maximum). In the course of engineering these strains, a number of mutations have been discovered in their adhE genes, which encode both alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. To understand the effects of these mutations, the adhE genes from six strains of C. thermocellum and T. saccharolyticum were cloned and expressed in Escherichia coli, the enzymes produced were purified by affinity chromatography, and enzyme activity was measured. In wild-type strains of both organisms, NADH was the preferred cofactor for both ALDH and ADH activities. In high-ethanol-producing (ethanologen) strains of T. saccharolyticum, both ALDH and ADH activities showed increased NADPH-linked activity. Interestingly, the AdhE protein of the ethanologenic strain of C. thermocellum has acquired high NADPH-linked ADH activity while maintaining NADH-linked ALDH and ADH activities at wild-type levels. When single amino acid mutations in AdhE that caused increased NADPH-linked ADH activity were introduced into C. thermocellum and T. saccharolyticum, ethanol production increased in both organisms. Structural analysis of the wild-type and mutant AdhE proteins was performed to provide explanations for the cofactor specificity change on a molecular level. This work describes the characterization of the AdhE enzyme from different strains of C. thermocellum and T. saccharolyticum. C. thermocellum and T. saccharolyticum are thermophilic anaerobes that have been engineered to make high yields of ethanol and can solubilize components of plant biomass and ferment the sugars to ethanol. In the course of engineering these strains, several mutations arose in the bifunctional ADH/ALDH protein AdhE, changing both enzyme activity and cofactor specificity. We show that changing AdhE cofactor specificity from mostly NADH linked to mostly NADPH linked resulted in higher ethanol production by C. thermocellum and T. saccharolyticum.

Experimentally Increased Codon Bias in the DrosophilaAdhGene Leads to an Increase in Larval, But Not Adult, Alcohol Dehydrogenase Activity

Although most amino acids can be encoded by more than one codon, the synonymous codons are not used with equal frequency. This phenomenon is known as codon bias and appears to be a universal feature of genomes. The translational selection hypothesis posits that the use of optimal codons, which match the most abundant species of isoaccepting tRNAs, results in increased translational efficiency and accuracy. Previous work demonstrated that the experimental reduction of codon bias in the Drosophila alcohol dehydrogenase (Adh) gene led to a significant decrease in ADH protein expression. In this study we performed the converse experiment: we replaced seven suboptimal leucine codons that occur naturally in the Drosophila melanogaster Adh gene with the optimal codon. We then compared the in vivo ADH activities imparted by the wild-type and mutant alleles. The introduction of optimal leucine codons led to an increase in ADH activity in third-instar larvae. In adult flies, however, the introduction of optimal codons led to a decrease in ADH activity. There is no evidence that other selectively constrained features of the Adh gene, or its rate of transcription, were altered by the synonymous replacements. These results are consistent with translational selection for codon bias being stronger in the larval stage and suggest that there may be a selective conflict over optimal codon usage between different developmental stages.

Isolation and characterization of a novel short-chain alcohol dehydrogenase gene from Panax ginseng

The cDNA of alcohol dehydrogenase (PgADH) was isolated and characterized from the leaf of Panax ginseng. The cDNA had an open reading frame of 801 bp and a deduced amino acid sequence of 266 residues. The calculated molecular mass of the mature protein is approximately 29 kDa with a predicated isoelectric point of 6.84. Homology analysis revealed that the deduced amino acid of PgADH shares a high degree of homology with the short-chain ADH proteins of other plants. Genomic DNA hybridization analysis indicated that PgADH represents a multi-gene family. The expression of PgADH under various environmental stresses was analyzed at different time points using real-time PCR. ABA, SA and especially JA (80-fold) significantly induced PgADH expression within 24 h of treatment. The positive responses of PgADH to abiotic stimuli suggest that ginseng ADH may protect against hormone-related environmental stresses.

TheCorynebacterium glutamicumNCgl2281 Gene Encoding an RNase E/G Family Endoribonuclease Can Complement theEscherichia coli rng::catMutation but Not therne-1Mutation

The Corynebacterium glutamicum NCgl2281 gene encodes an RNase E/G family endoribonuclease having an additional N-terminal domain of unknown function. In this study, we constructed plasmids expressing the full length (FL) and the N-terminally truncated form (DeltaN) of NCgl2281 and examined their complementation ability as to Escherichia coli rng::cat and rne-1 mutations. Both FL- and DeltaN-NCgl2281 rescued the defects caused by the rng::cat mutation, i.e., accumulation of 16S rRNA precursor, overproduction of the AdhE protein, and growth inhibition on M9 glucose medium. On the other hand, they did not complement the rne-1 mutation. These results indicate that the C. glutamicum NCgl2281 endoribonuclease is functionally more closely related to the E. coli RNase G than to RNase E.

Novel characteristics of UDP-glucose dehydrogenase activities in maize: non-involvement of alcohol dehydrogenases in cell wall polysaccharide biosynthesis

UDP-glucose dehydrogenase (UDPGDH) activity was detected in extracts of maize cell-cultures and developing leaves. The reaction product was confirmed as UDP-glucuronate. Leaf extracts from null mutants defective in one or both of the ethanol dehydrogenase genes, ADH1 and ADH2, had similar UDPGDH activities to wild-type, showing that UDPGDH activity is not primarily due to ADH proteins. The mutants showed no defect in their wall matrix pentose:galactose ratios, or matrix:cellulose ratio, showing that ADHs were not required for normal wall biosynthesis. The majority of maize leaf UDPGDH activity had K (m) (for UDP-glucose) 0.5-1.0 mM; there was also a minor activity with an unusually high K (m) of >50 mM. In extracts of cultured cells, kinetic data indicated at least three UDPGDHs, with K (m) values (for UDP-glucose) of roughly 0.027, 2.8 and >50 mM (designated enzymes E(L), E(M) and E(H) respectively). E(M) was the single major contributor to extractable UDPGDH activity when assayed at 0.6-9.0 mM UDP-Glc. Most studies, in other plant species, had reported only E(L)-like isoforms. Ethanol (100 mM) partially inhibited UDPGDH activity assayed at low, but not high, UDP-glucose concentrations, supporting the conclusion that at least E(H) activity is not due to ADH. At 30 microM UDP-glucose, 20-150 microM UDP-xylose inhibited UDPGDH activity, whereas 5-15 microM UDP-xylose promoted it. In conclusion, several very different UDPGDH isoenzymes contribute to UDP-glucuronate and hence wall matrix biosynthesis in maize, but ADHs are not responsible for these activities.

Gametophytic Variant of Alcohol Dehydrogenase ADH-P in Sugar Beet: Inheritance and Influence on Pseudo-Compatibility

A gametophytic variant ADH-P (alcohol dehydrogenase in pollen) was found in one plant in the sugar beet inbred line C257c. It was found only in developing and mature pollen, attempts failed to find it in other tissues. Rather than as an independent protein, ADH-P appeared as a variant of the ADH protein, accompanying the main proteins ADH1-FF and ADH1-SS. Therefore it can be suggested, that Adh-P is a gene-modifier decreasing the mobility of the basic protein Adhl in a manner similar for FF and SS isozymes. The native and modified proteins are enabled to form ADH1-F ADH-P and ADH1-S ADH-P heterodimers. The ADH-P is transferred on the female side as a Mendelian factor, on the male side with lowered frequency: 18% instead of the 50% expected. Independent inheritance was observed for Adh-P and Adhl, as well as for Adh-P and Idh2. Pseudo-compatibility was associated with Adh-P genotypes, 67% among Adh-P+ plants being self-incompatible, whereas among Adh-P- plants there was only 20%. A gametophytic variant of ADH-P is shown to decrease the adaptability of the male and, probably, the female gametophytes in sugar beet.

Anaerobic growth does not support biofilm formation in Escherichia coli K-12

Association with a surface in a structure known as biofilm is the prevailing microbial lifestyle. Here we show the kinetics of biofilm formation of Escherichia coli W3110 in static cultures growing under aerobic or anaerobic conditions. Aerobically growing cells in LB medium started to produce detectable amounts of biofilm after 4 to 8 h, displaying maximal accumulation of formed biofilm at 24 h, corresponding to the onset of stationary phase. Then an abrupt reduction in the biomass of the biofilm was observed. This decrease was not prevented by external addition of fresh nutrients and coincided with the depletion of oxygen as measured by the enzymatic activity of the AdhE protein. No biofilm formation was detected in cultures grown anaerobically in LB or LB supplemented with nitrate, nitrite, DMSO or fumarate, even after 72 h of incubation, well inside the stationary phase, suggesting that under anaerobic growth conditions E. coli cannot form biofilms.

Compensatory evolution of a precursor messenger RNA secondary structure in the Drosophila melanogaster Adh gene.

Evidence for the evolutionary maintenance of a hairpin structure possibly involved in intron processing had been found in intron 1 of the alcohol dehydrogenase gene (Adh) in diverse Drosophila species. In this study, the putative hairpin structure was evaluated systematically in Drosophila melanogaster by elimination of either side of the stem using site-directed mutagenesis. The effects of these mutations and the compensatory double mutant on intron splicing efficiency and ADH protein production were assayed in Drosophila melanogaster Schneider L2 cells and germ-line transformed adult flies. Mutations that disrupt the putative hairpin structure right upstream of the intron branch point were found to cause a significant reduction in both splicing efficiency and ADH protein production. In contrast, the compensatory double mutant that restores the putative hairpin structure was indistinguishable from the WT in both splicing efficiency and ADH level. It was also observed by mutational analysis that a more stable secondary structure (with a longer stem) in this intron decreases both splicing efficiency and ADH protein production. Implications for RNA secondary structure and intron evolution are discussed.

In vivo introduction of unpreferred synonymous codons into the Drosophila Adh gene results in reduced levels of ADH protein.

The evolution of codon bias, the unequal usage of synonymous codons, is thought to be due to natural selection for the use of preferred codons that match the most abundant species of isoaccepting tRNA, resulting in increased translational efficiency and accuracy. We examined this hypothesis by introducing 1, 6, and 10 unpreferred codons into the Drosophila alcohol dehydrogenase gene (Adh). We observed a significant decrease in ADH protein production with number of unpreferred codons, confirming the importance of natural selection as a mechanism leading to codon bias. We then used this empirical relationship to estimate the selection coefficient (s) against unpreferred synonymous mutations and found the value (s >or= 10(-5)) to be approximately one order of magnitude greater than previous estimates from population genetics theory. The observed differences in protein production appear to be too large to be consistent with current estimates of the strength of selection on synonymous sites in D. melanogaster.

Isolation and characterization of the genomic region from Drosophila kuntzei containing the Adh and Adhr genes.

The nucleotide sequences of the Adh and Adhr genes of Drosophila kuntzei were derived from combined overlapping sequences of clones isolated from a genomic library and from cloned PCR and inverse-PCR fragments. Only a proximal promoter was detected upstream of the Adh gene, indicating that D. kuntzei Adh is regulated by a one-promoter system. Further upstream of the Adh structural gene, an adult enhancer region (AAE) was found that contains most of the regulatory sequences described for AAEs of other Drosophila species. Analysis of the ADH protein showed an amino acid change from valine to threonine in the active site at position 189 which is also found in D. funebris but is otherwise unique among Drosophila. This difference alone may be responsible for the very low ADH activity found in this species and may cause a difference in substrate usage pattern. Codon bias in Adh and Adhr was comparable and found to be very low compared with other species. Phylogenetic analysis showed that D. kuntzei is closest related to D. funebris and D. immigrans. The time of divergence between D. kuntzei and D. funebris was estimated to be 14.2-20.2 Myr and that between D. kuntzei-D. funebris and D. immigrans to be 30.8-44.0 Myr. An analysis of the genetic variation in the Adh gene and upstream sequences of four European strains showed that this gene was highly variable. Overall nucleotide diversity (pi) was 0.0139, which is two times higher than that in D. melanogaster.

Extensive overproduction of the AdhE protein by rng mutations depends on mutations in the cra gene or in the Cra-box of the adhE promoter

Escherichia coli RNase G encoded by the rng gene is involved in degradation of adhE mRNA. Overproduction of the AdhE protein by rng mutants was found to depend on the genetic background of strains derived from DC272 ( adhC81) or MC1061. We found that DC272 carried a point mutation in the Cra-binding site of the adhE promoter. The Cra protein encoded by the cra gene is known to act as a repressor of adhE. P1-phage-mediated transduction and lacZ fusion analysis with the mutant adhE promoter confirmed that this mutation is responsible for overproduction. On the other hand, Southern hybridization revealed that MC1061 had a 0.85-kb deletion of the cra gene. Overproduction of AdhE in the MC1061 background was reversed to the wild-type levels by introduction of a plasmid carrying the cra + gene. These results indicated that expression of the adhE gene was regulated transcriptionally by Cra and posttranscriptionally by RNase G.