Alcohol Dehydrogenase Transcript Research Articles

Waterlogging-induced increase in fermentation and related gene expression in the root of cucumber (Cucumis sativus L.)

Waterlogging usually leads plant cells to shift their metabolism towards anaerobic fermentation pathways due to the hypoxia stress. The roles of two different levels of waterlogging treatments (water level was kept on the base of hypocotyls and on the base of the first true leaf) in induction of fermentation in waterlogged roots of cucumber line ‘Zaoer-N’ (waterlogging tolerant line) were examined. Root activities, numbers of dead cells, transcripts of genes encoding pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH), and activities of the PDC, ADH, and LDH enzymes, concentrations of acetaldehyde, alcohol and lactate were quantified during 48h of waterlogging, and 48h of subsequent recovery. The results showed that PDC, ADH, and LDH transcripts, activities of PDC, ADH and LDH, and concentrations of acetaldehyde, alcohol and lactate were upregulated or increased during waterlogging, and decreased with recovery time prolonged. The results revealed that waterlogging up to the base of first true leaf responded more severe than waterlogging up to the base of hypocotyls in root activity and numbers of dead cells, which were mainly caused by alcohol fermentation.

Gene transcript accumulation associated with physiological and chemical changes during developmental stages of strawberry cv. Camarosa

Association between molecular, physiological, and chemical changes during developmental stages of ‘Camarosa’ strawberry were investigated. Fruit were evaluated for flesh firmness, L-ascorbic acid, total phenolic content and volatile compounds, antioxidant capacity, and transcript accumulation of putative genes: expansin (Exp), pectate lyase (PL), pectin methylesterase (PME), polygalacturonase (PG), β-galactosidase (β-Gal), phenylalanine ammonia lyase (PAL), anthocyanidin synthase (ANS), L-galactose dehydrogenase (LGalDH) and l-galactono-1,4-lactone dehydrogenase (GLDH), alcohol dehydrogenase (ADH) and alcohol acetyl transferase (AAT). Flesh firmness reduction was correlated with an initial increase in Exp2 and Exp5, as well as PLa and PLb transcript accumulation, followed by increases in transcript accumulation of PME, PG, PLc, and β-Gal. A reduction in the antioxidant potential was associated with a decrease in ferulic acid, caffeic acid, (+)-catechin, (−)-epicatechin, quercetin and kaempferol content. ANS, LGalDH,GLDH, ADH and AAT transcripts, anthocyanins, total phenolics, gallic acid, ρ-hydroxybenzoic acid, ρ-coumaric acid and l-ascorbic acid, and ester volatiles increased as fruit matured.

Ethanolic Fermentation Enzymes, their Products and Transcription of Alcohol Dehydrogenase from Bell Pepper Fruit Held under Various Low Oxygen Atmospheres.

The concentrations of ethanol and acetaldehyde in bell pepper fruit were very low during storage at 1%, 3%, 5%, and 10% O2, and air at 20°C. At 0% O2, ethanol and acetaldehyde to a lesser extent, rapidly accumulated in its tissue. The activity of alcohol dehydrogenase (ADH) was about 10 times that of pyruvate decarboxylase (PDC) during storage. The ADH activity greatly increased in the fruit exposed to 1% and 3% O2, but in those exposed to 0%, 5%, and 10% O2 it was slightly higher than that of the control. The PDC activity greatly increased in the fruit exposed to 1%, 3%, and 5% O2, while those of fruits exposed to 0% and 10% O2 were slightly higher than that of the control. ADH transcripts in the fruit exposed to 5% and 10% O2 or air were not detectable, whereas those in the fruit exposed to 1% O2 were significantly higher than those in 0% and 3% O2 on day 1 at 20°C. The discrepancy of the enzyme activities and their product levels are discussed.

The class I alcohol dehydrogenase gene is glucocorticoid-responsive in the rat hepatoma microcell hybrid cell line, 11-3.

Expression of the class I alcohol dehydrogenase (ADH) gene in the rat hepatoma microcell hybrid cell line, 11-3, was examined. The steady-state level of ADH mRNA in 11-3 was approximately 2-fold higher than that or rat liver and Fao, the parental cell line of 11-3. Removal of steroid hormones by activated charcoal from the serum in which 11-3 cells were maintained resulted in a significant decrease in the level of ADH transcript. Dexamethasone at a concentration of 1 muM increased the ADH mRNA content in 11-3 in a time-dependent fashion, up to 48 hr after its addition to cells that had first been deprived of steroid hormones. In addition, levels of ADH transcript in cells treated with dexamethasone increased in a dose-dependent manner, and the concentration of dexamethasone required to achieve half-maximal activation was 5 nM. By using the techniques of reverse transcription and polymerase chain reaction, and by taking advantage of a restriction polymorphism present between the rat and mouse ADH cDNA, we found that 11-3 contained both the rat and mouse class I ADH transcripts, although the rat sequence accounted for the great majority. Moreover, levels of both rat and mouse class I ADH transcripts increased in a similarly time-dependent manner in cells treated with dexamethasone. These results indicate that expression of class I ADH gene in 11-3 is high and is regulated by glucocorticoids, making the cell line an excellent model for the in vitro study of ADH expression.

Cloning of the Mouse Class IV Alcohol Dehydrogenase (Retinol Dehydrogenase) cDNA and Tissue-specific Expression Patterns of the Murine ADH Gene Family

Humans possess five classes of alcohol dehydrogenase (ADH), including forms able to oxidize ethanol or formaldehyde as part of a defense mechanism, as well as forms acting as retinol dehydrogenases in the synthesis of the regulatory ligand retinoic acid. However, the mouse has previously been shown to possess only three forms of ADH. Hybridization analysis of mouse genomic DNA using cDNA probes specific for each of the five classes of human ADH has now indicated that mouse DNA cross-hybridizes to only classes I, III, and IV. With human class II or class V ADH cDNA probes, hybridization to mouse genomic DNA was very weak or undetectable, suggesting either a lack of these genes in the mouse or a high degree of mutational divergence relative to the human genes. cDNAs for murine ADH classes I and III have previously been cloned, and we now report the cloning of a full-length mouse class IV ADH cDNA. In Northern blot analyses, mouse class IV ADH mRNA was abundant in the stomach, eye, skin, and ovary, thus correlating with the expression pattern for the mouse Adh-3 gene previously determined by enzyme analysis. In situ hybridization studies on mouse stomach indicated that class IV ADH transcripts were abundant in the mucosal epithelium but absent from the muscular layer. Comparison of the expression patterns for all three mouse ADH genes indicated that class III was expressed ubiquitously, whereas classes I and IV were differentially expressed in an overlapping set of tissues that all contain a large component of epithelial cells. This expression pattern is consistent with the ability of classes I and IV to oxidize retinol for the synthesis of retinoic acid known to regulate epithelial cell differentiation. The results presented here indicate that the mouse has a simpler ADH gene family than the human but has conserved class IV ADH previously shown to be a very active retinol dehydrogenase in humans.

Cellular localization of the class I alcohol dehydrogenase transcript in adult rat tissues

The mammalian class I alcohol dehydrogenase is the principal enzyme responsible for ethanol metabolism. While it is regarded primarily as a liver-specific enzyme, class I alcohol dehydrogenase is known to be present in a number of extrahepatic tissues. The purpose of the current study is to define the tissue and cellular distribution of the dehydrogenase transcript in four rat tissues previously shown to contain high levels of mRNA: the liver, the proximal small intestine, the colon and the testis. Localization of the transcript was examined in formalin-fixed, paraffin-embedded rat tissues by in situ hybridization using radioactively labelled antisense rat alcohol dehydrogenase RNA probe. In the liver, the dehydrogenase message is localized primarily to the perivenous hepatocytes. In the proximal small intestine and the colon, the message follows a vertical gradient of distribution along the crypt-villus and the crypt-surface epithelium axes, respectively, with the base of the crypt exhibiting the greatest concentration. In the testis, the message is localized primarily to cells in the interstitium. These findings illustrate a highly compartmentalized nature of distribution of the class I alcohol dehydrogenase transcript in the tissues studied and may help to elucidate the metabolic functions of this enzyme in these tissues.

Expression of the class I alcohol dehydrogenase gene in developing rat fetuses.

Class I alcohol dehydrogenase (ADH) is the principal enzyme responsible for ethanol oxidation in mammals. Although primarily regarded as an enzyme that functions in the adult, Class I ADH has been reported to be present in fetal tissues. By in situ hybridization, we demonstrated the tissue localization of the Class I ADH transcript in developing rat fetuses between Days 15 (E15) and 18 (E18) of gestation. Abundant transcripts were present in epidermis, lung, and urinary bladder. In these tissues, the messages were localized primarily to the superficial layer of the epithelium and increased with development. The liver exhibited significant signals only in the E18 fetus, when parenchymal hepatocytes first appeared. The E15 and E16 small intestines, with their epithelium arranged in a stratified fashion, displayed signals in the submucosal mesenchymal layer. By E17, a rearrangement of the intestinal epithelium into an almost monolayer configuration was observed. This change was associated with a redistribution of the ADH transcript to the surface of the epithelium. Further relocation of the messages was noted in the adult small intestine, in which they became concentrated in the base of the crypt. These findings indicate that expression of the rat class I ADH gene follows a dynamic course in specific epithelial tissues during fetal development. In addition, the apparent superficial localization of the ADH message in most of these tissues suggests that ADH functions in metabolizing either endogenously or exogenously derived alcohol substrates present in the fetal environment.

Ethanolic fermentation in transgenic tobacco expressing Zymomonas mobilis pyruvate decarboxylase.

During oxygen limitation in higher plants, energy metabolism switches from respiration to fermentation. As part of this anaerobic response the expression of genes encoding pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) is strongly induced. In addition there is ample evidence for post-translational regulation. In order to understand this multi-level regulation of the anaerobic response, we provided tobacco with the constitutive capacity of ethanolic fermentation by expressing a PDC gene derived from the obligate anaerobe Zymomonas mobilis. The protein accumulated to high levels and was active in an in vitro assay. During the first 2-4 h of anoxia, acetaldehyde accumulated to 10- to 35-fold and ethanol to 8- to 20-fold higher levels than in wild-type. Under normoxic conditions no accumulation of acetaldehyde and ethanol could be measured. Instead, the two products may be immediately re-metabolized in tobacco leaf tissue. We show that aerobic fermentation takes place when the respiratory system is inhibited. Although these conditions enhance ethanolic fermentation under normoxia, they fail to increase ADH transcript levels. These results indicate that anaerobic transcription is triggered not by the metabolic consequences of oxygen limitation, but directly through an oxygen-sensing system.

Estradiol regulates class I alcohol dehydrogenase gene expression in renal medulla of male rats by a post-transcriptional mechanism

Rat kidney contains alcohol dehydrogenase (ADH) activity which appears to be identical to the class I ADH expressed in liver. Treatment of male rats with estradiol for 10 days induced ADH activity and protein in the kidney approximately 3-fold. This was not the result of suppression of testosterone levels by estrogen, as castration did not increase ADH activity. In situ hybridization of kidney sections showed that ADH transcripts were localized to the medulla, that the basal level of mRNA is very low in the male, and that the induction of ADH mRNA by estradiol was approximately 10-fold. As estimated from Northern blot analysis, the induction of the mRNA was approximately 7-fold. Thus, induction of ADH mRNA substantially exceeded the increase in ADH activity and protein. Since the estradiol-treated rats lost weight relative to the oil-injected controls, the effect of starvation on ADH mRNA in kidney was examined. Starvation decreased kidney ADH activity by about 30% but increased mRNA about 2-fold. Time course experiments demonstrated induction of ADH mRNA by estradiol within 1 h with the maximum level achieved by 24 h. The transcription rate of the ADH gene as assessed by nuclear run-on assays performed at 1 and 24 h after treatment with estradiol was unchanged. We conclude that estradiol induces ADH mRNA in kidney by a post-transcriptional mechanism.

Molecular Characterization of the Soybean Alcohol Dehydrogenase Gene Family Amplified in Vitro by the Polymerase Chain Reaction

Soybean (Glycine max) alcohol dehydrogenase (ADH) cDNAs were amplified in vitro from total RNA by the polymerase chain reaction (PCR). The amplification strategy involved first strand cDNA synthesis from anaerobic cotyledon total RNA using an 18-thymidine primer. The second strand cDNA primer was a conserved sequence near the 5' end of known plant ADH transcripts. The PCR products were ligated into a plasmid vector and unique clones were isolated on the basis of size and restriction pattern. Sequence analysis revealed three distinct classes of soybean ADH cDNAs, all of which showed high homology to Adh genes from maize and peas. RNA blot hybridization analyses showed differential expression patterns for these genes. One gene, expressed constitutively in all seedling organs, was inducible by anaerobiosis, one gene was expressed only in anaerobic organs, and the third gene was expressed predominantly in anaerobic roots.

Structure and expression of the rat class I alcohol dehydrogenase gene

Clones containing the rat class I alcohol dehydrogenase (ADH) gene were isolated from a Charon 4A genomic library. The gene spans approximately 13 kb and comprises nine exons and eight introns. The upstream 436 bp contain canonical TATA and CCAAT sequences, and inverted CACCC box, a TG3 box found in mouse and human ADH promoters, and regions of homology to glucocorticoid response elements. The 5′-untranslated region of the ADH transcript has the potential to form a stable stem-loop structure. The first intron contains an unusual stretch of alternating purines and pyrimidines similar to that found in the same location in the mouse ADH gene. The amino acid insertion found in rat alcohol dehydrogenase results from a shift in the 3′ splice junction of the fourth intron which adds an extra three base pairs to the fifth exon. Intron-exon boundaries are otherwise identical to those in mouse and human ADH genes. H4IIE cells stably transfected with plasmids containing the chloramphenicol acetyltransferase (CAT) gene fused behind the first 436 bp of the promoter region express CAT, but the CAT activity is not inducible by dexamethasone. The elements responsible for glucocorticoid stimulation of ADH gene transcription appear to reside outside of this region.

Cloned Drosophila alcohol dehydrogenase genes are correctly expressed after transfection into Drosophila cells in culture.

We have obtained correct transcription of the cloned alcohol dehydrogenase (Adh) gene of Drosophila melanogaster after DNA-mediated gene transfer into Drosophila cells in culture. Supercoiled plasmids, each containing various regions of the Adh gene cloned in pBR327, were introduced into Schneider line 2 (SL2) cells by the calcium phosphate-DNA transfection technique. Although these cells do not normally express their endogenous Adh genes, they do express the exogenous genes as shown by primer extension and nuclease S1 analyses of RNA isolated 48 hr after transfection. The resulting alcohol dehydrogenase (ADH) transcripts, both the larval and adult types, have the correct 5' ends and are properly spliced. The transfected cells have also acquired ADH enzyme activity. The levels of enzyme activity and of ADH protein crossreacting material in cells transfected with different Adh plasmids correlate directly with the level of ADH transcripts. When a mutant Adh gene cloned from an ADH-negative mutant fly with a defect in the splicing of ADH RNA is transfected into the Schneider line 2 cells, the resulting ADH RNA is not spliced properly and there is no synthesis of ADH; thus, the mutant gene transfection into cell culture mimics the mutant phenotypes observed in the mutant fly.