Encoding Alcohol Dehydrogenase Research Articles

Molecular analysis of mouse alcohol dehy drogenase: nucleotide sequence of the Adh-1 gene and genetic mapping of a related nucleotide sequence to chromosome 3

The mouse has three genes ( Adh) encoding alcohol dehydrogenase (ADH) enzymes of different tissue specificity and catalytic properties. Identified regulatory loci are known to affect the expression of Adh-1 and Adh-3, which are closely linked on chromosome 3. The Adh-1 gene product is expressed predominantly in liver, and its mRNA product is androgen-inducible in kidney. In this study, genomic clones of Adh-1 were obtained from a Balb/cJ DNA library. The nucleotide sequences of all exons, intron/exon boundaries and 5′- and 3′-flanking regions were obtained. The gene spans nearly 13 kbandis divided into nine exons and eight introns. The transcription start point of this gene was determined by Sl nuclease mapping studies and presumptive regulatory regions in the 5′ -flanking regions were identified, including a TATA box and a glucocorticoid-responsive element. A restriction fragment length polymorphism in the Adh-1 gene was identified among inbred strains and mapped at the [ Adh-1,Adh-3] complex on chromosome 3. An additional ‘ Adh-Mke’ sequence in the genome was also mapped to chromosome 3 approx. 9 centiMorgans from Adh-1.

Production of α-Amylase by Yeast

The enzyme alpha-amylase confers to an organism the enzymatic activity for the degradation of polyglucosides with alpha-1,4 glycosidic bonds such as starch and glycogen which are among the major storage compounds in plants and animals. Most alpha-amylases are single polypeptides of molecular weights around 50,000 dalton. They are generally found in the digestive tract of animals and in germinating seeds. Among the products released upon enzymatic degradation of polyglucosides maltose, a sugar that can be utilized as carbon source by yeast, is a major constituent. A cDNA segment complementary to mouse salivary amylase messenger RNA has been inserted into the yeast expression vector pMA56 behind the promoter of the gene encoding alcohol dehydrogenase I of yeast. Yeast transformants harboring plasmids with the normal orientation of the promoter and the mouse amylase cDNA gene produce amylase and release the enzyme in free form into the culture medium. Approximately 90% of the amylase activity is found in the medium. Yeast strains carrying MAL allele and transformed with a plasmid which directed the synthesis of mouse alpha-amylase were tested on plates containing starch and in batch fermentations using different high molecular weight sugars and oligosaccharides as carbon source. The results of these experimentsmore » will be discussed. (Refs. 21).« less

Molecular cloning and DNA sequence of the Arabidopsis thaliana alcohol dehydrogenase gene.

Arabidopsis thaliana provides an excellent experimental plant system for molecular genetics because of its remarkably small genome size, near absence of dispersed middle repetitive DNA, and short life cycle. We have cloned and determined the nucleotide sequence of a single-copy gene from A. thaliana likely to be the gene encoding alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1). The gene was isolated from a random recombinant library by cross-hybridization with a maize Adh1 gene probe. The DNA sequence contains an open reading frame capable of encoding a polypeptide the same length as maize ADH1 and ADH2 (379 amino acids) and having approximately equal to 80% homology with both maize enzymes. This open reading frame is interrupted by six introns whose positions are conserved with six of the nine intron positions present in both maize genes. The 5' and 3' untranslated regions are, respectively, 58 and 204 base pairs long. Sequences important for eukaryotic gene expression such as the TATA box, polyadenylylation signal, and intron splicesite sequences are found in the expected locations. The gene hybridizes to a specific anaerobically induced RNA in Arabidopsis whose appearance correlates with the anaerobic induction of Arabidopsis ADH protein.

Interferon-induced guanylate-binding proteins: mapping of the murine Gbp-1 locus to chromosome 3.

GBP-1 is the predominant species of a family of guanylate-binding proteins synthesized in mouse cells in response to interferons (IFNs) alpha, beta, or gamma. IFN inducibility of this 65,000-Da protein is controlled by alleles at a single autosomal locus, Gbp-1, with allele a encoding inducibility and allele b noninducibility. Here, we present evidence suggesting that both alleles occur in outbred populations of wild mice. Using recombinant inbred strains and classical linkage analysis of offspring of two-point and three-point backcrosses we demonstrate that Gbp-1 is linked to Adh-3 (encoding alcohol dehydrogenase C2) and VaJ (varitintwaddler-Jackson) located on the distal part of chromosome 3. The relevant recombination frequencies (RFs) (+/- SE) were 3.5 (+/- 1.1) and 11.7 (+/- 2.8)%, respectively. We further show that strain B6.C-H-23c/By(HW 53), congenic for a small segment of chromosome 3, carries the BALB/c alleles at both the Gbp-1 and the Adh-3 locus and not the alleles of the B6 background strain confirming the chromosomal location and close linkage of the two loci.

Purification and molecular properties of mouse alcohol dehydrogenase isozymes.

Alcohol dehydrogenase isozymes from mouse liver (A2 and B2) and stomach (C2) tissues have been purified to homogeneity using triazine-dye affinity chromatography. The enzymes are dimers with similar but distinct subunit sizes, as determined by SDS/polyacrylamide gel electrophoresis: A, 43000; B, 39000, and C, 47000. Zinc analyses and 1,10-phenanthroline inhibition studies indicated that the A and C subunits each contained two atoms of zinc, with at least one being involved catalytically, whereas the B subunit probably contained a single non-catalytic zinc atom. The isozymes exhibited widely divergent kinetic characteristics. A2 exhibited a Km value for ethanol of 0.15 mM and a broad substrate specificity, with Km values decreasing dramatically with an increase in chain length; C2 also exhibited this broad specificity for alcohols but showed a Km value of 232 mM for ethanol. These isozymes also showed broad substrate specificities as aldehyde reductases. In contrast, B2 showed no detectable activity as an aldehyde reductase for the aldehydes examined, and used ethanol as substrate only at very high concentrations (greater than 0.5 M). The isozyme exhibited low Km and high Vmax values, however, with medium-chain alcohols. Immunological studies showed that A2 was immunologically distinct from the B2 and C2 isozymes. In vitro molecular hybridization studies gave no evidence for association between the alcohol dehydrogenase subunits. The results confirm genetic analyses [Holmes, Albanese, Whitehead and Duley (1981) J. Exp. Zool. 215, 151-157] which are consistent with at least three structural genes encoding alcohol dehydrogenase in the mouse and confirm the role of the major liver isozyme (A2) in ethanol metabolism.

Biochemical genetics of aldehyde reductase in the mouse: Ahr-1--a new locus linked to the alcohol dehydrogenase gene complex on chromosome 3.

Electrophoretic and activity variants for a liver aldehyde reductase (AHR-A2) among strains of Mus musculus have been used in genetic analyses to demonstrate close linkage between the locus encoding this enzyme (designated Ahr-1) and the alcohol dehydrogenase gene complex on chromosome 3. No recombinants were observed between Adh-3 (encoding alcohol dehydrogenase C2; ADH-C2) and Ahr-1 among 42 backcross animals. Moreover, linkage disequilibrium between these loci was observed among 58 of 60 strains of mice examined and among seven recombinant inbred strains derived from C57BL/6J and BALB/c mice. Liver hexonate dehydrogenase (HDH-A) was electrophoretically invariant among the strains examined. Gel filtration analyses demonstrated that AHR-A2 and HDH-A had native molecular weights of approximately 80,000 and 32,000, respectively. Three-banded allozyme patterns for AHR-A2 in CBA/H x castaneus hybrid animals were consistent with a dimeric subunit structure. Comparative substrate and coenzyme specificities for AHR-A2, HDH-A, and ADH-A2 (liver ADH isozyme) were examined. AHR-A2 exhibited a defined specificity toward p-nitrobenzaldehyde as substrate, whereas the other enzymes exhibited broad specificities toward various aliphatic, aromatic, and monosaccharide aldehydes. It is proposed that Ahr-1 is a product of a gene duplication event during mammalian evolution of the primordial mammalian Adh locus and that considerable divergence in catalytic properties has subsequently occurred.

CDNA cloning and induction of the alcohol dehydrogenase gene ( Adh1 ) of maize

cDNA clones of Adh1, one of two genes encoding alcohol dehydrogenase (ADH; alcohol:NAD(+) oxidoreductase, EC 1.1.1.1) in the maize genome, have been isolated. They were derived from mRNA extracted from anaerobically treated roots of maize seedlings. Identification was initially made on the basis of molecular weight and electrophoretic properties of the in vitro polypeptide obtained in hybridization-release-translation experiments. The identification was confirmed by antibody precipitation and by the use of maize stocks having different genetic constitutions at the Adh1 locus. The sequence of the longest cDNA segment, approximately 900 base pairs, was determined and appears to code for 168 COOH-terminal amino acids and to have a 3' nontranslated region of 364 base pairs. Reverse Southern hybridizations established that two different Adh1-S stocks produce a mRNA of 1,650 nucleotides, whereas an additional mRNA of 1,750 nucleotides is produced in three Adh1-F stocks. A 50-fold increase in Adh1 mRNA level occurs during anaerobiosis, reaching a maximum at 5 hr. Return to aerobic conditions indicates a half-life of more than 18 hr for the anaerobically induced Adh1 mRNA.