Dehydrogenase cDNA Research Articles

Human xanthine dehydrogenase cDNA sequence and protein in an atypical case of type I xanthinuria in comparison with normal subjects

To investigate the properties of xanthine dehydrogenase/xanthine oxidase (XDH/XO) deficiency in a patient with atypical type I xanthinuria, as indicated by oxypurine data, a cDNA sequence encoding XDH, XDH/XO immunoblot analysis and a competitive PCR assay were performed, and the results were compared with those of normal subjects. The xanthine dehydrogenase cDNA sequence of the patient was consistent with the controls, while immunologically reactive 150 kD XDH/XO protein was not present in the xanthinuric duodenal mucosa, unlike the control duodenal mucosa. In addition, a decrease in XDH/XO messenger RNA was found by competitive PCR. These results suggest that atypical type I xanthinuria is due to a decrease in messenger RNA of XDH/XO. Furthermore, it was considered that this decrease could explain the normal plasma level and near normal urinary excretion of hypoxanthine seen in this case of xanthinuria, though XDH/XO activity and protein were not detected spectrophotometrically and immunologically, respectively.

Site-directed mutagenesis studies of bovine liver cytosolic dihydrodiol dehydrogenase: the role of Asp-50, Tyr-55, Lys-84, His-117, Cys-145 and Cys-193 in enzymatic activity

A previous report on the cloning, bacterial expression and purification of bovine liver cytosolic dihydrodiol dehydrogenase (DD3) cDNA (1,330 bp in full length) using pKK223-3 expression vector characterize the properties of the recombinant DD3 in the aspects of substrate specificity and inhibitor sensitivity (Terada et al., Adv. Exp. Biol. Res. 414 (1997) 543–53). The nucleotide sequence of this DD3 cDNA completely matches that of bovine liver-type prostaglandin F synthase (PGFS) (Suzuki et al., J. Biol. Chem. 274 (1999) 241–8). In the present study, a large amount of recombinant DD3 (rDD3) was expressed in Escherichia coli BL21 (DE3) with a pET28a expression vector. The recombinant DD3 (rDD3) was easily and quickly purified to an apparent homogeneity with one step column chromatography of Ni 2+-affinity resin. The rDD3 showed essentially the same substrate specificity and inhibitor sensitivity as purified liver DD3 (DD3). To analyze the role of amino acid residues of DD3 in its enzymatic activity, site-directed mutagenesis of DD3 with PCR method was performed. The results of the analyses of these mutants in the aspects of substrate specificity and cofactor-binding suggested a variety of functions in the enzymatic activity: as an active site Tyr-55 may act as a general acid and Asp-50, Lys-84 and His-117 may play an important role in the control of protonation of Tyr-55 as a general acid in the dehydrogenase activity under higher pH conditions, though these residues may not be involved in reductase activity under lower pH conditions. Though the mutated DD3s (Cys to Ser) did not show significant differences in their substrate specificities, these mutants showed different sensitivities to SH-reagents. Present results indicate that Cys-193 may play an important role in the modulation of enzymatic activity under redox conditions generated with GSH+GSSG among five cysteines in DD3.

Isolation and expression of a cinnamyl alcohol dehydrogenase cDNA from perennial ryegrass (Lolium perenne)

A perennial ryegrass (Lolium perenne L.) cDNA library was screened with a PCR-amplified cad DNA fragment generated from ryegrass cDNA template using degenerate oligonucleotide primers. A full-length cDNA (LpeCad1) was isolated and confirmed to encode a cinnamyl alcohol dehydrogenase (CAD) enzyme by expression of activity in Escherichia coli. The recombinant enzyme catalyses conversion of coniferaldehyde and sinapaldehyde with similar efficiency, and apparent K m values below 10 µM were determined for these substrates, whereas weak substrate inhibition occurs above this concentration. The predicted perennial ryegrass CAD was very similar (88–87percnt; amino acid sequence identity) to the only other monocotyledonous plant CAD sequences available, those of maize and sugarcane, respectively. Southern blot hybridization analysis indicated that there may be two or three cad genes, or alleles, in perennial ryegrass. The ryegrass LpeCad1 gene resembles the maize cadgene in showing strong expression in root and stem tissues, but is also expressed at lower levels in shoot, leaf sheath, leaf blade and floral tissues.

Genetic control of HDL levels and composition in an interspecific mouse cross (CAST/Ei × C57BL/6J)

Strain CAST/Ei (CAST) mice exhibit unusually low levels of high density lipoproteins (HDL) as compared with most other strains of mice, including C57BL/6J (B6). This appears to be due in part to a functional deficiency of lecithin:cholesterol acyltransferase (LCAT). LCAT mRNA expression in CAST mice is normal, but the mice exhibit several characteristics consistent with functional deficiency. First, the activity and mass of LCAT in plasma and in HDL of CAST mice were reduced significantly. Second, the HDL of CAST mice were relatively poor in phospholipids and cholesteryl esters, but rich in free cholesterol and apolipoprotein A-I (apoA-I). Third, the adrenals of CAST mice were depleted of cholesteryl esters, a phenotype similar to that observed in LCAT- and acyl-CoA:cholesterol acyltransferase-deficient mice. Fourth, in common with LCAT-deficient mice, CAST mice contained triglyceride-rich lipoproteins with "panhandle"-like protrusions. To examine the genetic bases of these differences, we studied HDL lipid levels in an intercross between strain CAST and the common laboratory strain B6 on a low fat, chow diet as well as a high fat, atherogenic diet. HDL levels exhibited complex inheritance, as 12 quantitative trait loci with significant or suggestive likelihood of observed data scores were identified. Several of the loci occurred over plausible candidate genes and these were investigated. The results indicate that the functional LCAT deficiency is unlikely to be due to variations of the LCAT gene. Our results suggest that novel genes are likely to be important in the control of HDL metabolism, and they provide evidence of genetic factors influencing the interaction of LCAT with HDL.

Mannitol-1-phosphate dehydrogenase from Cryptococcus neoformans is a zinc-containing long-chain alcohol/polyol dehydrogenase.

Cryptococcus neoformans, the causative agent of cryptococcosis, produces large amounts of mannitol in culture and in infected mammalian hosts. Although there is considerable indirect evidence that mannitol synthesis may be required for wild-type stress tolerance and virulence in C. neoformans, this hypothesis has not been tested directly. It has been proposed that mannitol-1-phosphate dehydrogenase (MPD) is required for fungal mannitol synthesis, but no MPD-deficient fungal mutants or cDNAs or genes encoding fungal MPDs have been described. Therefore, C. neoformans was purified from a 148 kDa homotetramer of 36 kDa subunits that catalysed the reaction mannitol1-phosphate+NAD--><--fructose 6-phosphate+NADH. Partial peptide sequences were used to isolate the corresponding cDNA and gene, and the deduced MPD protein was found to be homologous to the zinc-containing long-chain alcohol/polyol dehydrogenases. Lysates of Saccharomyces cerevisiae transformed with the cDNA of interest (but not vector-transformed controls) contained MPD catalytic activity. Lastly, Northern analyses demonstrated MPD mRNA in glucose- and mannitol-grown C. neoformans cells. Thus, MPD has been purified and characterized from C. neoformans, and the corresponding cDNA and gene (MPD1) cloned and sequenced. Availability of C. neoformans MPD1 should permit direct testing of the hypotheses that (i) MPD is required for mannitol biosynthesis and (ii) the ability to synthesize mannitol is essential for wild-type stress tolerance and virulence.

Cloning and characterization of glyceraldehyde-3-phosphate dehydrogenase cDNA of Japanese flounder Paralichthys olivaceus +

SUMMARY: Japanese flounder Paralichthys olivaceus glyceraldehyde-3-phosphate dehydrogenase (GAPDH or G3PD) cDNA was cloned and sequenced. Japanese flounder GAPDH was found to consist of 333 amino acid residues and to exhibit a high degree of homology to previously reported GAPDH of vertebrates, in both the DNA and amino acid sequences. Curiously, Japanese flounder GAPDH was found to be more homologous to GAPDH of Xenopus, chicken and mammals than to that of rainbow trout. We developed primers for RT-PCR and used this method to amplify GAPDH mRNA from various tissues to determine whether it could be used as an internal standard. The GAPDH was expressed in all organs and tissues, and confirmed that it represents a good internal standard positive control for the study of gene expression.

Quantification of relative mRNA expression in the rat brain using simple RT-PCR and ethidium bromide staining

We developed a protocol for quantification of relative gene expression using reverse transcription-polymerase chain reaction (RT-PCR) without the use of radioisotopes, special equipment or extra nucleotide fragments, such as competitors. The relative gene expression of GABA A receptor β 1 subunit (GABA ARβ 1) and phospholipase C β 4 subtype (PLCβ 4) in rat cerebrum and cerebellum were determined by comparing the ratio of PCR products generated by linear amplification of the target cDNA segments and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA segment as a reference. The density of PCR products was measured from digitized images of photographs of ethidium-bromide-stained agarose gels. The linear region of PCR amplification was within the linear range (from 0.3 to 12 ng DNA in a single band) of the detection system. The accuracy of the present method was <2-fold difference in gene expression in a single determination and a 1.5-fold difference was statistically significant after repeated measurements. The estimated relative expression of PLCβ 4 was significantly higher in cerebellum than cerebrum, and that of GABA ARβ 1 was the same in these two regions. Using the present method, it is possible to quantify several different subunits and subtypes of known ion channel, neurotransmitter receptor and intracellular signaling enzyme gene families.

Analysis of a ferric leghemoglobin reductase from cowpea ( Vigna unguiculata) root nodules

Ferric leghemoglobin reductase (FLbR), an enzyme reducing ferric leghemoglobin (Lb) to ferrous Lb, was purified from cowpea ( Vigna unguiculata) root nodules by sequential chromatography on hydroxylapatite followed by Mono-Q HR5/5 FPLC and Sephacryl S-200 gel filtration. The purified cowpea FLbR had a specific activity of 216 nmol Lb 2+O 2 formed min −1 mg −1 of enzyme for cowpea Lb 3+ and a specific activity of 184 nmol Lb 2+O 2 formed min −1 mg −1 of enzyme for soybean Lb 3+. A cDNA clone of cowpea FLbR was obtained by screening a cowpea root nodule cDNA library. The nucleotide sequence of cowpea FLbR cDNA exhibited about 88% similarity with soybean ( Glycine max) FLbR and 85% with pea ( Pisum sativum) dihydrolipoamide dehydrogenase (DLDH, EC 1.8.1.4) cDNAs. Conserved regions for the FAD-binding site, NAD(P)H-binding site, and disulfide active site were identified among the deduced amino acid sequences of cowpea FLbR, soybean FLbR, pea DLDH and other enzymes in the family of the pyridine nucleotide-disulfide oxido-reductases.

Cloning of two glutamate dehydrogenase cDNAs from Asparagus officinalis: sequence analysis and evolutionary implications.

Two different amplification products, termed c1 and c2, showing a high similarity to glutamate dehydrogenase sequences from plants, were obtained from Asparagus officinalis using two degenerated primers and RT-PCR (reverse transcriptase polymerase chain reaction). The genes corresponding to these cDNA clones were designated aspGDHA and aspGDHB. Screening of a cDNA library resulted in the isolation of cDNA clones for aspGDHB only. Analysis of the deduced amino acid (aa) sequence from the full-length cDNA suggests that the gene product contains all regions associated with metabolic function of NAD glutamate dehydrogenase (NAD-GDH). A first phylogenetic analysis including only GDHs from plants suggested that the two GDH genes of A. officinalis arose by an ancient duplication event, pre-dating the divergence of monocots and dicots. Codon usage analysis showed a bias towards A/T ending codons. This tendency is likely due to the biased nucleotide composition of the asparagus genome, rather than to the translational selection for specific codons. Using principal coordinate analysis, the evolutionary relatedness of plant GDHs with homologous sequences from a large spectrum of organisms was investigated. The results showed a closer affinity of plant GDHs to GDHs of thermophilic archaebacterial and eubacterial species, when compared to those of unicellular eukaryotic fungi. Sequence analysis at specific amino acid signatures, known to affect the thermal stability of GDH, and assays of enzyme activity at non-physiological temperatures, showed a greater adaptation to heat-stress conditions for the asparagus and tobacco enzymes compared with the Saccharomyces cerevisiae enzyme.

Cloning and Characterization of a Putative Human d-2-Hydroxyacid Dehydrogenase in Chromosome 9q

There is little information on d-isomer-specific dehydrogenases in humans. Identification of d-2-hydroxyglutaric aciduria, an inherited metabolic disorder associated with severe neurological dysfunction, highlights the role of d-isomers in human metabolism. The possibility of a defect in d-2-hydroxyglutarate dehydrogenation prompted us to employ E. colid-2-hydroxyacid dehydrogenase cDNA to search the human expressed sequence tags database. Two human EST homologues were retrieved and sequenced. Analysis showed the two clones were identical with 1258 nucleotides encoding 248 amino acids of the putative human d-2-hydroxyacid dehydrogenase. It was highly homologous to bacterial d-2-hydroxyacid dehydrogenases (46%), d-phosphoglycerate dehydrogenase (38%), and formate dehydrogenase (36%) at the amino acid level. The gene is expressed ubiquitously in tissue, most abundantly in liver, and was mapped to chromosome 9q between markers WI-3028 and WI-93330. To our knowledge this is the first cloning and characterization of the cDNA for a human d-isomer specific NAD+-dependent 2-hydroxyacid dehydrogenase.

Cloning of two glutamate dehydrogenase cDNAs from &lt;i&gt;Asparagus officinalis&lt;/i&gt;: Sequence analysis and evolutionary implications

Cloning of two glutamate dehydrogenase cDNAs from &lt;i&gt;Asparagus officinalis&lt;/i&gt;: Sequence analysis and evolutionary implications

Biochemistry of glutaric aciduria type I: activities of in vitro expressed wild-type and mutant cDNA encoding human glutaryl-CoA dehydrogenase.

Glutaric aciduria type I (GA I; McKusick 231670) is caused by deficiency of glutaryl-CoA dehydrogenase activity (EC 1.3.99.7), a key enzyme in the catabolism of the amino acids lysine, hydroxylysine and tryptophan (Besrat et al 1969; Goodman et al 1975). The failure to metabolize glutaryl-CoA may give rise to elevated levels of glutarate, 3-hydroxyglutarate and glutaconate in the body fluids of the patient. Recent studies showed the leading role of 3-hydroxyglutarate in the pathogenesis of GA I (Baric et al, unpublished). Yet the source of this dicarboxylic acid is not well understood. Glutaryl-CoA dehydrogenase catalyses the reaction in two steps: dehydrogenation of glutaryl-CoA to glutaconyl-CoA and decarboxylation of glutaconyl-CoA to crotonyl-CoA, which can be measured separately (Hartel et al 1993). Whereas the hydration of glutaconyl-CoA to 3-hydroxyglutarylCoA is catalysed by methylglutaconase (EC 4.2.1.18; Liesert and Buckel, unpublished), the formation of glutaconyl-CoA is less clear. It could arise from glutaryl-CoA dehydrogenase mutants in which the decarboxylase activity is more affected than the dehydrogenase activity. Another possibility would be a nonspecific oxidation of glutaryl-CoA by medium-chain acyl-CoA dehydrogenase (MCAD, EC 1.3.99.3) or similar enzymes. In order to address this question, mechanistically and clinically interesting mutants were introduced into a modified human glutaryl-CoA dehydrogenase cDNA that can be expressed in Escherichia coli.

Hes1 and Hes5 as notch effectors in mammalian neuronal differentiation.

While the transmembrane protein Notch plays an important role in various aspects of development, and diseases including tumors and neurological disorders, the intracellular pathway of mammalian Notch remains very elusive. To understand the intracellular pathway of mammalian Notch, the role of the bHLH genes Hes1 and Hes5 (mammalian hairy and Enhancer-of-split homologues) was examined by retrovirally misexpressing the constitutively active form of Notch (caNotch) in neural precursor cells prepared from wild-type, Hes1-null, Hes5-null and Hes1-Hes5 double-null mouse embryos. We found that caNotch, which induced the endogenous Hes1 and Hes5 expression, inhibited neuronal differentiation in the wild-type, Hes1-null and Hes5-null background, but not in the Hes1-Hes5 double-null background. These results demonstrate that Hes1 and Hes5 are essential Notch effectors in regulation of mammalian neuronal differentiation.

Activity of human 11-cis-retinol dehydrogenase (Rdh5) with steroids and retinoids and expression of its mRNA in extra-ocular human tissue

This report describes the activity of recombinant human Rdh5 (11-cis-retinol dehydrogenase) with steroids and retinoids and expression of the Rdh5 mRNA in extra-ocular human tissue. The data show that Rdh5 catalyses 9-cis-retinol metabolism equally efficiently as 11-cis-retinol metabolism and recognizes 5alpha-androstan-3alpha,17beta-diol and androsterone as substrates (3alpha-hydroxysteroid dehydrogenase activity), but not testosterone, dihydrotestosterone, oestradiol and corticosterone (lack of 17beta-hydroxysteroid and 11beta-hydroxysteroid dehydrogenase activities). Rdh5 mRNA expression was widespread in extra-ocular tissues with human liver (100% relative expression in extra-ocular tissues only) and mammary gland (97% relative to liver) showing the most intense signals. Other noteworthy relatively intense expression sites included colon (45%), thymus (43%), small intestine (39%), kidney (37%), bladder (29%), pancreas and spleen (28% each), heart (26%), uterus and ovary (25% each), testis (22%) and spinal cord (24%). Human fetal tissues also expressed Rdh5 with fetal liver showing the most intense expression among the fetal tissues (20%). Considered along with the identical nucleotide sequences in the untranslated regions of human Rdh5 and human 9-cis-retinol dehydrogenase cDNAs and the nearly identical nucleotide sequences overall (99% identity), the current results suggest that the two cDNAs represent a single gene product.

Cloning and Expression of Two Carbonyl Reductase-like 20β-Hydroxysteroid Dehydrogenase cDNAs in Ovarian Follicles of Rainbow Trout (Oncorhynchus mykiss)

In salmonid fish, 20β-hydroxysteroid dehydrogenase (20β-HSD) is a key enzyme involved in the production of oocyte maturation-inducing hormone (MIH), 17α,20β-dihydroxy-4-pregnen-3-one. Here we report the isolation of two cDNAs which encode proteins with high homology to carbonyl reductase-like 20β-HSD (CR/20β-HSD) from rainbow trout (Oncorhynchus mykiss) ovarian follicles. Genomic DNA analysis showed that the two CR/20β-HSD cDNAs are derived from two different genes. Northern blot and RT PCR analysis demonstrated that trout CR/20β-HSDs are broadly expressed in various tissues. Enzymatic characterization using recombinant CR/20β-HSD proteins produced inE. colishowed that the product of one of the two cDNAs had both 20β-HSD and CR activity, but the other had neither activity. Although the functional significance of the two genes remains unresolved, these results clearly demonstrate the presence of two distinct CR/20β-HSD transcripts in the trout ovary.

Cloning of Mouse 20.ALPHA.-Hydroxysteroid Dehydrogenase cDNA and Its mRNA Localization during Pregnancy.

20α-Hydroxysteroid dehydrogenase (20α-HSD) converts progesterone to a biologically inactive steroid, 20α-dihydroprogesterone. To examine a potential role of 20α-HSD in tissues other than corpus luteum (CL) during pregnancy, mouse 20α-HSD cDNA was cloned and was used for the examination of its mRNA localization in the conceptus and uterus at different stages of gestation. Using probes based on the rat sequence, mouse 20α-HSD cDNA clone was isolated from a cDNA library prepared from ovaries of day 12 pseudopregnant mice. The mouse 20α-HSD cDNA was 1193 bp in length and encoded 323 amino acids with an estimated molecular weight of 37 kD. The mouse 20α-HSD was homologous to the rat 20α-HSD in nucleotide and amino acid sequences (both in 93% homology), and to other members of aldo-keto reductase superfamily at the cofactor (NADP(H)) binding site. By in situ hybridization, 20α-HSD mRNA was localized in endometrial epithelial cells on days 10, 15 and 18 of pregnancy, maternal placental endothelial cells on day 10, and fetal epidermal cells on day 15. During pseudopregnancy as well as early pregnancy, however, 20α-HSD mRNA was not detected in endometrial cells. Together, 20α-HSD mRNA was localized in cells that surround fetuses during the mid and late stages of gestation.

Characterization of an aldehyde dehydrogenase gene fragment from mung bean (Vigna radiata) using the polymerase chain reaction.

NAD(P)+-dependent aldehyde dehydrogenases of higher plants have not been studied well enough at the moment as compared to those of mammals. The betaine aldehyde dehydrogenase gene was obtained from rice (Nacamyra et al., 1997). There is some information on the structure of spinach, sugar beet, sorghum, burley cDNAs (Rhodes and Hanson, 1993; Wood et al., 1996), non-phOsphorylating glyceraldehyde 3-phosphate dehydrogenase cDNAs from pea and maize (Habenicht et al., 1994), maize RF2 protein cDNA (about 60% identity to mammalian mitochondrial aldehyde dehydrogenases) (Cui et al., 1996), tobacco mitochondrial aldehyde dehydrogenase cDNA (op den Camp and Kuhlemeier, 1997).KeywordsAldehyde DehydrogenaseBetaine Aldehyde DehydrogenaseAldehyde Dehydrogenase GeneMitochondrial Aldehyde DehydrogenaseBetaine Aldehyde Dehydrogenase GeneThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Molecular characterisation and expression of a wound-inducible cDNA encoding a novel cinnamyl-alcohol dehydrogenase enzyme in lucerne (Medicago sativa L.)

A lucerne (alfalfa, Medicago sativa) stem cDNA library was screened with a cinnamyl-alcohol dehydrogenase (CAD) cDNA probe from tobacco (Nicotiana tabacum cv. Samsun). Two distinctly different cDNA clones (54% identical) were isolated and identified as putative CAD-encoding cDNAs by comparison of their nucleotide sequences with those of CAD-encoding DNA sequences from other plant species. One of the cDNAs, MsaCad2, was found to be 99.4% identical at the nucleotide level to the previously isolated lucerne cad cDNA which encodes a CAD isoform involved in lignin biosynthesis. The other cDNA, MsaCad1, has not been reported previously in lucerne, and encodes a protein related to the ELI3 class of elicitor-inducible defence-related plant proteins. The MsaCad1- and MsaCad2-encoded proteins were expressed in Escherichia coli and CAD1 was shown to be active with a range of cinnamyl, benzyl and aliphatic aldehyde substrates, while CAD2 was specific for the cinnamyl aldehydes only. Each of the respective genes is present as one or two copies. The MsaCad1 gene is expressed most actively in stem and floral tissue, whereas MsaCad2 is most actively expressed in stem, hypocotyl and root tissue. In stem tissue, expression of both genes occurs predominantly in internodes 4 and 5 (from the apex). MsaCad2, in contrast to MsaCad1, is not significantly expressed in the top three internodes of the stem. Both MsaCad1 and MsaCad2 are wound-inducible, and the wound-responsiveness of each gene is modulated by salicylic acid.

A sequence similar to bacterial transposable IS elements present in the 5' untranslated region of the bovine butanediol dehydrogenase cDNA.

A sequence similar to prokaryotic transposable elements was identified in the long 5' untranslated region (5'UTR) of the butanediol dehydrogenase cDNA isolated from a bovine brain lambda gt11 library. Several observations suggested that this sequence could be related to bacterial IS elements: (a) 58% nucleotide sequence identity, (b) 56% amino acid sequence identity, and (c) the presence of inverted terminal repeats. However, nucleotide sequence analyses of the 5'UTR bovine cDNA showed the presence of chain-terminating nucleotide substitutions that would render it incapable of encoding a functional transposase. Finally, it was observed that different vertebrate genomes have sequences related to this putative transposable element.

Cloning and characterization of NADP-mannitol dehydrogenase cDNA from the button mushroom, Agaricus bisporus, and its expression in response to NaCl stress.

Mannitol, a six-carbon sugar alcohol, is the main storage carbon in the button mushroom, Agaricus bisporus. Given the physiological importance of mannitol metabolism in growth, fruit body development, and salt tolerance of A. bisporus, the enzyme responsible for mannitol biosynthesis, NADP-dependent mannitol dehydrogenase (MtDH) (EC 1.1.1.138), was purified to homogeneity, and MtDH cDNA was cloned, sequenced, and characterized. To our knowledge, this represents the first report on the isolation of a cDNA encoding an NADP-dependent mannitol dehydrogenase. The MtDH cDNA contains an open reading frame of 789 bp encoding a protein of approximately 28 kDa. The N-terminal and internal amino acid sequences of the deduced protein exactly matched the ones determined from the purified MtDH subunit, whereas the amino acid composition of the deduced protein was nearly identical to that of the purified MtDH. The MtDH cDNA showed high homology with a plant-induced short-chain dehydrogenase from Uromyces fabae. Phylogenetic analysis based on amino acid sequences from mannitol(-1-phosphate) dehydrogenases indicated a close relationship between the substrate specificity of the enzymes and phylogenetic differentiation. Salt-stressed fruit bodies showed an overall increase in mannitol biosynthesis, as was evident from the increase in MtDH activity, MtDH abundance, and MtDH RNA accumulation. Furthermore, the MtDH transcript level seems to be under developmental control, as MtDH RNA accumulated during maturation of the fruit body.