Synthesis Of Alcohol Dehydrogenase Research Articles

Psammophytes Alyssum desertorum Stapf and Secale sylvestre Host Are Sensitive to Soil Flooding

While morphological and functional traits enable hydrophytes to survive under waterlogging and partial or complete submergence, the data on responses of psammophytes—sand plants—to flooding are very limited. We analyzed the effect of 5- and 10-day soil flooding on the photosynthetic apparatus and the synthesis of alcohol dehydrogenase (ADH), heat shock proteins 70 (HSP70), and ethylene in seedlings of psammophytes Alyssum desertorum and Secale sylvestre using electron microscopy, chlorophyll a fluorescence induction, and biochemical methods. It was found that seedlings growing under soil flooding differed from those growing in stationary conditions with such traits as chloroplast ultrastructure, pigment content, chlorophyll fluorescence induction, and the dynamics of ADH, HSP, and ethylene synthesis. Although flooding caused no apparent damage to the photosynthetic apparatus in all the variants, a significant decrease in total photosynthesis efficiency was observed in both studied plants, as indicated by decreased values of φR0 and PIABS,total. More noticeable upregulation of ADH in S. sylvestre, as well as increasing HSP70 level and more intensive ethylene emission in A. desertorum, indicate species-specific differences in these traits in response to short-term soil flooding. Meanwhile, the absence of systemic anaerobic metabolic adaptation to prolonged hypoxia causes plant death.

Assessment of alcohol dehydrogenase synthesis and aerenchyma formation in the tolerance of Sium L. species (Apiaceae) to water-logging

To survive and reproduce under the conditions of limited oxygen supply, plants develop morphological and biochemical features that are either constitutive or induced by flooding. We performed a comparative study of the root cortex structure and alcohol dehydrogenase (ADH) synthesis in widespread aerial-aquatic Sium latifolium and terrestrial S. sisaroideum (Apiaceae) in response to changes in the soil water content to check the hypothesis that plasticity of structural and metabolic traits may ensure survival and adaptation of these species at variable soil moisture − from aerated soil to water-logging. Plant grown under natural and experimental conditions were used for microscopy and enzyme assays. We show that the soil moisture determined changes in the root cortex structure and systemic switching-on of anaerobic fermentation in the investigated species. Water-logging of the terrestrial species S. sisaroideum induced first ADH synthesis and then development of new aerenchymatous adventitious roots that are inherent for aerial-aquatic plants of S. latifolium. Histochemical localization showed high ADH activity in the root apices. The aerenchyma in adventitious roots of both species consisted of longitudinal gas spaces in the inner cortex and enlarged intercellular spaces of a “honeycomb” type in the outer cortex. Thus, both Sium species showed complex anaerobic biochemical and structural responses to flooding, promoting the internal aeration of roots. The high plasticity of the root systems in S. latifolium and S. sisaroideum have led to their widespread distribution in nature.

Breeding for submergence tolerance in rice (Oryza sativa L.) and its management for flash flood in rainfed low land area: A review

For rainfed low land paddy ecosystems which is supposed to withstand the sudden rise of water level for short period of time i.e. up to two weeks has been considered as the submergence tolerant. Proper screening and evaluation method are very important for cost effective and economic breeding procedures that will benefits the farmers. The agro morphological characters which are responsible for submergence tolerance; character association and path coefficient analysis will be very much useful to select them for further assessment. Hybrid rice will be another approach which could mitigate the flash flood constraints. Proper understanding of anatomical characters such as aerenchyma formation and changes of diameter of aerenchyma of root, shoot and leaf will be very much important for submergence tolerance if it is linked with genetic factors. Biochemical manipulation of different plant hormone such as gibberellic acid, abscisic acid and ethylene; enzyme like alcohol dehydrogenase synthesis; chlorophyll content and non structural carbohydrate content of shoot has significant role for enhancement of survival during underwater growth. Plant extracts or herbal crude product in the form of dry and wet treatments along with proper growth nutrients will be a holistic approach for sustainable and integrated agriculture and will be a tool for crop improvement in future breeding programme for submergence tolerance in rice.

Синтез білка теплового шоку HSP70 і алкогольдегідрогенази у листках Arabidopsis Thaliana і Sium Sisaroideum у відповідь на затоплення ґрунту

Синтез білка теплового шоку HSP70 і алкогольдегідрогенази у листках Arabidopsis Thaliana і Sium Sisaroideum у відповідь на затоплення ґрунту

Construction of a butyrate-producing E. coli strain without the use of heterologous genes

Multistage construction of an E. coli strain containing no foreign genes which is capable of producing butyrate has been carried out. At the first stage, deletions in gene fadR encoding a protein repressor of an operon for fatty acid degradation and gene aceF responsible for the synthesis of pyruvate dehydrogenase were introduced in the strain MG1655 genome. Then, a mutant obtained from the above strain by induced mutagenesis and capable of growth on ethanol as a sole carbon source under aerobic conditions was selected. It was shown that growth of the mutant on ethanol is provided by two mutations. One of them (a substitution: 257G → A) is located in the regulatory region of gene adhE that controls the synthesis of alcohol-dehydrogenase; the other, containing a substitution Glu568 → Lys, affects the structural portion of the gene. As a result of the consequent mutagenesis of the obtained strain and selection on indicating media, variants capable of growing on butyrate and butanol as sole carbon sources and putatively bearing mutations in gene atoC (encoding transcriptional activator of atoDAB operon) were selected. At the last stage of the work, gene atoB, encoding the synthesis of the thiolase II enzyme, was placed under the control of a constitutive promoter Ptet, and the functional allele of gene aceF was introduced. The resulting E. coli strain (ΔfadR, adhE, atoC, Ptet-atoB) accumulates 800 mg/l of butyrate upon growth on glucose-containing medium under semi-anaerobic (oxygen limited) conditions. Introduction of an additional deletion in gene ldhA encoding lactate dehydrogenase in the strain genome leads to a further growth of a butyrate production up to 1.3 g/l.

Effect of leptin on liver alcohol dehydrogenase

The effect of leptin on liver alcohol dehydrogenase (ADH) was determined in male rats. Administration of one or three daily doses of leptin (1 μg/g of body weight intraperitoneally) increased ADH activity. Leptin enhanced ADH synthesis without an effect on ADH degradation. Leptin did not change ADH mRNA, indicating that the effect of leptin in enhancing ADH occurs at the post-transcriptional level. Leptin increased eukaryotic initiation factor (eIF) 2α, eIF2B activity, and the eIF4E–eIF4G complex, while it decreased the inhibitory complex of eIF4E with the eIF4E-binding protein-1 (4E-BP1). Leptin increased mammalian target of rapamycin (mTor) that phosphorylates 4E-BP1. In conclusion, leptin increases liver ADH activity and ADH protein due to an increase in synthesis which occurs at the post-transcriptional level. The effect of leptin in enhancing translational initiating factors may be of significance in the regulation not only of ADH but also of many other proteins.

Molecular characteristics and transcription of the gene encoding a multifunctional alcohol dehydrogenase in relation to the deactivation of pyruvate formate-lyase in the ruminal bacterium Streptococcus bovis.

To clarify the deactivation mechanism of pyruvate formate-lyase (PFL) and its role in the regulation of fermentation in Streptococcus bovis, the molecular properties and genetic expression of multifunctional alcohol dehydrogenase (ADHE) were investigated. S. bovis was found to have ADHE, which was deduced to consist of 872 amino acids with a molecular mass of 97.4 kDa. The ADHE was shown to harbor three enzyme activities: (1) alcohol dehydrogenase, (2) coenzyme-A-linked acetaldehyde dehydrogenase that catalyzes the conversion of acetyl-CoA to ethanol, and (3) PFL deactivase. Similar to Escherichia coli ADHE, S. bovis ADHE required Fe2+ for its activity. The gene encoding ADHE ( adhE) was shown to be monocistronic. The level of adhE mRNA changed in parallel with the mRNA levels of the genes encoding PFL (pfl) and PFL-activating enzyme (act) as the growth conditions changed, although these genes are independently transcribed. Synthesis of ADHE, PFL-activating enzyme, and PFL appears to be regulated concomitantly. Overexpression of ADHE did not cause a change in the formate-to-lactate ratio. It is conceivable that ADHE is not significantly involved in the reversible inactivation of active PFL under anoxic conditions. Partition of the flow from pyruvate appears to be mainly regulated by the activities of lactate dehydrogenase and PFL.

Protein Synthesis During Oxygen Deprivation in Cotton

The alteration of protein synthesis induced by oxygen deprivation has been examined in the root tips of cotton (Gossypium hirsutum cv. Siokra), a plant that is intolerant to anoxia. Using [35S]methionine labelling and two-dimensional gel electrophoresis it was demonstrated that 14 major polypeptides are being selectively synthesised during oxygen deprivation. These polypeptides have been designated the cotton anaerobic polypeptides (ANPs), and have estimated molecular masses that correspond to molecular masses of ANPs from other plant species. However, compared to maize, several of the major molecular weight classes are absent, suggesting that the response to oxygen deprivation in cotton is simpler than that of maize. Alcohol dehydrogenase (ADH) activity is induced by oxygen deprivation. Using western analysis we have determined that this increase in activity is correlated with the accumulation of the ADH polypeptide and that three of the major cotton ANPs are ADH, including the most intensely labelled ANP, demonstrating that the synthesis of ADH constitutes a major part of the response in cotton.

Hyperbolic growth of Thermoanaerobacter thermohydrosulfuricus (Clostridium thermohydrosulfuricum) increases ethanol production in pH-controlled batch culture

Thermoanaerobacter thermohydrosulfuricus Rt8.B1 exhibited hyperbolic growth (i.e. a continuous rate of growth, without diauxie, during growth and utilization of two carbon sources) on mixed carbohydrate substrates when grown in pH-controlled batch culture. Hyperbolic growth was observed with xylose in combination with either glucose or cellobiose. Diauxic growth ways observed when T. thermohydrosulfuricus Rt8.B1 was grown on a glucose plus cellobiose substrate mix. The major fermentation end-products under all substrate conditions were ethanol and acetate. Ethanol production varied depending on the substrate supplied and was always greatest on mixtures that included xylose (i.e. hyperbolic growth). High ethanol-to-acetate ratios could not be explained on the basis of a greater substrate uptake and thus more ethanol production under these conditions, or by variations in the levels of acetate kinase and NADP-linked alcohol dehydrogenase synthesis. The high ethanol-to-acetate ratio could not be increased by growing T.thermohydrosulfuricus Rt8.B1 under a partial pressure of hydrogen (1 atm) or by growth at different pH. Growth under these conditions decreased the ethanol-to-acetate ratio.

Concurrent Synthesis and Degradation of Alcohol Dehydrogenase in Elicitor-Treated and Wounded Potato Tubers

The accumulation of alcohol dehydrogenase (ADH) in arachidonic acid-elicited potato (Solanum tuberosum L.) tuber discs was studied. In accordance with our previous report of the accumulation of Adh mRNA beginning 2 hours after elicitor treatment (DP Matton, CP Constabel, N Brisson [1990] Plant Mol Biol 14: 775-783), immunoprecipitation of ADH from in vivo labeled discs indicated that ADH synthesis occurred as early as 12 hours after treatment. However, levels of ADH activity and protein, as shown by enzyme assay and immunoblot, did not rise in parallel but decreased during the first 24 hours of treatment. After 24 hours, ADH activity and protein began to increase, reaching a several-fold increase at 96 hours after elicitation. Water-treated control discs showed a similar though delayed and less pronounced pattern. These results imply a turnover of ADH following elicitor treatment of potato tuber discs. As shown by nondenaturing gel electrophoresis, the synthesis and degradation involved the same ADH isozyme.

Evidence Does Not Support Ethylene as a Cue for Synthesis of Alcohol Dehydrogenase and Pyruvate Decarboxylase During Exposure to Hypoxia

Low O2 concentrations increased the activity of alcohol dehydrogenase (ADH; EC 1.1.1.1) and pyruvate decarboxylase (PDC; EC 4.1.1.1), but exogenously applied ethylene did not reproduce this effect in either maize roots or rice coleoptiles. However, ethylene was clearly exerting its known effects on the maize roots, since it inhibited root extension, and promoted aerenchyma formation. Furthermore, treatment with silver nitrate, an antagonist of ethylene action, failed to inhibit the rise in ADH activity in O2-deficient maize roots. We conclude that ethylene causes morphological adaptations to O2-deficiency, but is not involved in concurrent increases in activities of ADH and PDC. Alternative causes for the increases in enzyme activity are proposed.

Cloning and characterization of the gene for a methanol-utilising alcohol dehydrogenase from Bacillus stearothermophilus

The cloning and characterization of the alcohol dehydrogenase (ADH) gene ( adh) from Bacillus stearothermophilus strain DSM2334, an obligate aerobe, are described. The clone directed the synthesis of ADH as judged on Western blots, activity gels and tetrazolium plates. It specified an enzyme that oxidised methanol as well as ethanol. The enzyme was found to be encoded by a single gene in B. stearothermophilus which did not cross-hybridise to adh clones from Escherichia coli, yeast or maize. The cloned gene was expressed in E. coli but activity was not detected in Bacillus subtilis, despite stable maintenance of the recombinant plasmid in this host. The gene is catabolite-repressed in DSM2334.

Molecular consequences of two formaldehyde-induced mutations in the alcohol dehydrogenase gene ofDrosophila melanogaster

Adhfn23 and Adhfn24 are two formaldehyde-induced, homozygous-viable, alcohol dehydrogenase-null mutants that bear lesions in the gene that codes for the alcohol dehydrogenase (ADH; EC 1.1.1.1) of Drosophila melanogaster. Adhfn23 contains a 34-base pair deletion in the C-terminal coding region of the alcohol dehydrogenase structural gene. By immunological and molecular analysis, we show that the deletion shifts the translation reading frame and results in a prematurely truncated polypeptide product (10 amino acids shorter than wild type) that cross-reacts with antibody raised against ADH. The steady-state level of alcohol dehydrogenase mRNA present in this mutant is close (97%) to that in the wild type, but the steady-state level of alcohol dehydrogenase-like protein is 50% lower. Moreover, the rate of alcohol dehydrogenase synthesis in Adhfn23 flies is reduced to 60% of that found in the wild type. Hence both the rate of synthesis and the rate of degradation of alcohol dehydrogenase are affected. In contrast, Adhfn24 which contains an 11-base pair deletion in the N-terminal coding region of the ADH gene, synthesizes no immunodetectable protein, and the amount of alcohol dehydrogenase mRNA is less than half that of wild-type flies. As with Adhfn23, the deletion in Adhfn24 results in a change in the reading frame. Unlike Adhfn23, however, nucleic acid sequence data indicate that polypeptide chain elongation can proceed for a considerable distance (over 130 amino acids) beyond the deletion. Based upon antigenic binding-site predictions, the resultant aberrant protein (projected 195 amino acids in length) would share few antigenic sites with the alcohol dehydrogenase from the wild type, which may account for the lack of immunoprecipitable material in this mutant. The contrasting effects these two deletions have on the Drosophila ADH mRNA levels and ADH protein levels are discussed.

UGA nonsense mutation in the alcohol dehydrogenase gene of Drosophila melanogaster

A mutant gene, which we have designated Adh nB , codes for a defective form of the enzyme alcohol dehydrogenase in Drosophila melanogaster. We show that the polypeptide encoded by Adh nB is approximately 2000 M r smaller than the protein synthesized under the direction of the wild-type alcohol dehydrogenase gene. In contrast, the alcohol dehydrogenase mRNA produced by both genes is the same size. We cloned and sequenced a portion of the protein-coding region of Adh nB and compared it to the same region in the wild-type gene. We found a single base substitution: a change of the TGG tryptophan codon at amino acid 235 to a TGA termination codon. This nonsense mutation accounts for the observed reduction in size of the alcohol dehydrogenase polypeptide. In further studies, we found that the steady-state levels of alcohol dehydrogenase mRNA in flies carrying the Adh nB gene and the wild-type alcohol dehydrogenase gene were indistinguishable. However, the steady-state level of alcohol dehydrogenase polypeptide was reduced to 1% of wild-type levels in flies with the Adh nB gene. Moreover, the rate of alcohol dehydrogenase synthesis in mutant flies was reduced to 50% of that found in wild type. The aberration in Adh nB thus affects both the rate of synthesis and the rate of degradation of the alcohol dehydrogenase peptide. Adh nB is the first reported nonsense mutant in Drosophila.

Alcohol dehydrogenase in Arabidopsis: analysis of the induction phenomenon in plantlets and tissue cultures

Alcohol dehydrogenase (ADH) activity is expressed in Arabidopsis seeds and tissue cultures. During the germination process, ADH activity declines rapidly and is no longer detectable in 9- to 10-day-old seedlings. The synthesis of ADH could be demonstrated in seedlings submitted to anaerobiosis by 35S-methionine incorporation studies.

Onset of Alcohol Dehydrogenase Synthesis during Microsporogenesis in Maize

During male gametophyte development the synthesis of several proteins occurs from transcripts of the haploid genome. Alcohol dehydrogenase (ADH1), a developmentally regulated protein, was chosen for study to determine the stage at which its synthesis was initiated and the pattern of its synthesis during microsporogenesis. The ability of ADH to reduce p-nitro blue tetrazolium chloride in situ was used as an indicator of enzyme activity. Maize strains heterozygous for adh1 were utilized to provide an internal control, 50% of the grains being adh1(+) and 50% being adh1(-). No ADH activity was detectable when tetrads were first formed after meiosis. Activity was initially detected soon after the tetrads began to break apart but before the microspores in the tetrads had completely separated. The transcription of the adh1 gene from the haploid genome must thus occur very soon after meiosis is completed. ADH activity increases at a constant rate thereafter until microspore mitosis when an increase in the rate takes place which lasts until generative cell division. Thereafter, there is a marked decrease in the rate of accumulation of ADH activity.

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.

Genetic determination of the developmental program for maize scutellar alcohol dehydrogenase: Involvement of a recessive, trans‐acting, temporal‐regulatory gene

AbstractThe developmental program of alcohol dehydrogenase (ADH) activity in the scutellum of maize strain R6‐67 is different from that of W64A. The level of scutellar ADH activity in R6‐67 remains relatively high during the course of early sporophytic development as compared to the commonly observed pattern. In the typical inbred strain W64A, the activity of ADH declines substantially during that period. The variance values from the crosses between R6‐67 and W64A reveal that the trait is under genetic control. Detailed genetic analysis suggests that a single gene is responsible for the altered developmental program of ADH activity in R6‐67. This gene meets the criteria for temporal regulatory genes and is different from Adh2, the structural gene which codes the ADH‐2 isozyme. We have designated this gene as Adr1 (alcohol dehydrogenase regulator, #1). Adr1 is unlinked to Adh2. There is no de novo synthesis of ADH in the scutellum during germination, and the difference in the activity level reflects the difference in the amount of enzyme protein as demonstrated by density labeling and rocket immunoelectrophoresis. Thus, it appears that Adr1 may regulate the degradation of ADH.

Selective synthesis of alcohol dehydrogenase during anaerobic treatment of maize

Anaerobic treatment of a maize seedling mediates the synthesis of five major, native proteins and seven polypeptide size-classes; slab polyacrylamide and autoradiographic techniques were used to analyze extracts from single primary roots. The alcohol dehydrogenase-1 polypeptide is most dramatically synthesized and accumulated during anaerobiosis, as compared to aerobic control data. Allozymes were used to identify alcohol dehydrogenase unequivocally. Our results pertain to interpretations of previous studies on the alcohol dehydrogenase gene system in maize, and to work on the stress proteins of Drosophila.

Proteinkristalle und Tubuli-Bündel in Hefezellen IV. Biochemische und elektronenmikroskopische Untersuchungen zur Induktion von Kristallen aus Alkoholdehydrogenase (ADH)

Cells of Saccharomyces carlsbergensis H 60 synthesize alcohol dehydrogenase (ADH) in media containing 2% lactat, 1% ethanol or 0.1% glucose. Crystals may be induced in protoplasts of these cells. Increase of glucose concentration in the medium results in diminished ADH synthesis and decreased tendency for crystal formation. Repression of ADH synthesis by glucose results in the formation of a protein (MG 110000 D), the significance of which is discussed. Early stages of crystal formation inside the cell are demonstrated electronmicroscopically. At first dense material accumulates between opposite membranes of neighbouring mitochondria. Within mitochondria frequently membrane bundles occur in close vicinity to crystals. These ADH-crystals arise from this material.