Dehydrogenase cDNA Research Articles

Sequence analysis of a mannitol dehydrogenase cDNA from plants reveals a function for the pathogenesis-related protein ELI3.

Mannitol is the most abundant sugar alcohol in nature, occurring in bacteria, fungi, lichens, and many species of vascular plants. Celery (Apium graveolens L.), a plant that forms mannitol photosynthetically, has high photosynthetic rates thought to results from intrinsic differences in the biosynthesis of hexitols vs. sugars. Celery also exhibits high salt tolerance due to the function of mannitol as an osmoprotectant. A mannitol catabolic enzyme that oxidizes mannitol to mannose (mannitol dehydrogenase, MTD) has been identified. In celery plants, MTD activity and tissue mannitol concentration are inversely related. MTD provides the initial step by which translocated mannitol is committed to central metabolism and, by regulating mannitol pool size, is important in regulating salt tolerance at the cellular level. We have now isolated, sequenced, and characterized a Mtd cDNA from celery. Analyses showed that Mtd RNA was more abundant in cells grown on mannitol and less abundant in salt-stressed cells. A protein database search revealed that the previously described ELI3 pathogenesis-related proteins from parsley and Arabidopsis are MTDs. Treatment of celery cells with salicylic acid resulted in increased MTD activity and RNA. Increased MTD activity results in an increased ability to utilize mannitol. Among other effects, this may provide an additional source of carbon and energy for response to pathogen attack. These responses of the primary enzyme controlling mannitol pool size reflect the importance of mannitol metabolism in plant responses to divergent types of environmental stress.

Substrate Specificity, Gene Structure, and Tissue-specific Distribution of Multiple Human 3α-Hydroxysteroid Dehydrogenases

We have expressed in Escherichia coli functionally active proteins encoded by two human cDNAs that were isolated previously by using rat 3 alpha-hydroxysteroid dehydrogenase cDNA as the probe. The expressed proteins catalyzed the interconversion between 5 alpha-dihydrotestosterone and 5 alpha-androstane-3 alpha,17 beta-diol. Therefore, we name these two enzymes type I and type II 3 alpha-hydroxysteroid dehydrogenases. The type I enzyme has a high affinity for dihydrotestosterone, whereas the type II enzyme has a low affinity for the substrate. The tissue-specific distribution of these two enzymes was determined by reverse transcription polymerase chain reaction using gene-specific oligonucleotide primers. The mRNA transcript of the type I enzyme was found only in the liver, whereas that of the type II enzyme appeared in the brain, kidney, liver, lung, placenta, and testis. The structure and sequence of the genes encoding these two 3 alpha-hydroxysteroid dehydrogenases were determined by analysis of genomic clones that were isolated from a lambda EMBL3 SP6/T7 library. The genes coding for the type I and type II enzymes were found to span approximately 20 and 16 kilobase pairs, respectively, and to consist of 9 exons of the same sizes and boundaries. The exons range in size from 77 to 223 base pairs (bp), whereas the introns range in size from 375 bp to approximately 6 kilobase pairs. The type I gene contains a TATA box that is located 27 bp upstream of multiple transcription start sites. In contrast, the type II gene contains two tandem AP2 sequences juxtaposed to a single transcription start site.

Human glucose-6-phosphate dehydrogenase Lysine 205 is dispensable for substrate binding but essential for catalysis

By site-directed mutagenesis of the cloned human glucose-6-phosphate dehydrogenase cDNA, lysine 205 (the residue that after reacting with pyridoxal-5′-phosphate renders inactive enzyme) was mutated to threonine (K205T) to remove the amino group, or to arginine (K205R) to displace the position of the amino group, in order to analyze the role of its nucleophilic group in position ϵ. Compared to the wild-type enzyme, the K205T and K205R mutants retain a specific activity of 2.6 and 11.4%, respectively; their catalytic specificity ( K cat/ K m) is drastically decreased, whereas the K m values for both substrates are only slightly increased. These findings in the light of the 3D structure of G6PD suggest that the ϵ-amino group of lysine 205 can favour a hydrogen bond within the active pocket essential for catalysis.

Cloning, sequencing, and determination of the sites of expression of mouse sorbitol dehydrogenase cDNA.

Sorbitol dehydrogenase is one of the enzymes in the polyol pathway, which is thought to be implicated in the pathogenesis of diabetic complications. The cDNA encoding mouse sorbitol dehydrogenase was cloned from a liver library and its sequence was determined. The open reading frame encodes a product of 356 amino acids that shares high similarity with the human and rat liver sorbitol dehydrogenases (83% and 93% identity, respectively). The 3'-untranslated region contains a truncated L1Md repeat element inserted in reverse relative to the sorbitol dehydrogenase cDNA. Northern-blot hybridization showed that the testis has the highest level of expression, followed by kidney, liver, and lung. Low levels of expression were also observed in lens, brain, and skeletal muscle. In situ hybridization revealed that in the kidney, the highest concentration of sorbitol dehydrogenase mRNA is observed in the cortex, but is absent from the inner medulla. The parenchymal cells of the liver showed strong expression while the cells of the hepatic vasculature did not hybridize. The sorbitol dehydrogenase expression in the seminiferous tubules was mostly associated with the mature cells of the developing germ cells, confirming the usefulness of sorbitol dehydrogenase as an enzyme indicator for sexual maturation. The seminal vesicle, where most of the seminal fructose is produced, also showed a high level of expression in the epithelial cells. The mouse sorbitol dehydrogenase cDNA will be useful in the studies of the involvement of the polyol pathway in diabetic complications.

Molecular evidence that diploid Stylosanthes humilis and diploid Stylosanthes hamata are progenitors of allotetraploid Stylosanthes hamata cv. Verano

Restriction fragment length polymorphism analysis, using peroxidase, O-methyltransferase, phenylalanine ammonia-lyase, and coniferyl alcohol dehydrogenase cDNAs isolated from Stylosanthes humilis, as probes, provided molecular evidence for the genetic origin of the naturally occuring allotetraploid genotype Stylosanthes hamata cv. Verano (2n = 4x = 40). Hybridization patterns strongly suggest that the likely progenitors of S. hamata cv. Verano were a diploid S. humilis (2n = 2x = 20) and a diploid S. hamata (2n = 2x = 20) species.

Direct expression of the pig testicular 3 alpha/beta(20 beta)- hydroxysteroid dehydrogenase cDNA using baculovirus expression system

The direct expression of the pig testicular 3 alpha/beta(20 beta)- hydroxysteroid dehydrogenase (HSD) cDNA using a baculovirus expression system was investigated. A minimum essential cDNA containing the coding region of 3 alpha/beta(20 beta)-HSD was amplified by polymerase chain reaction (PCR) to extend the DNA linker including the Bam HI restriction site in the both ends of the cDNA. As the template, 3 alpha/beta(20 beta)-HSD cDNA, pBS 52, which was subcloned to Bluescript II was used. The PCR fragment was ligated to Bam HI-cut transfer plasmid (pBlueBac III). Recombinant transfer plasmid (pBlueBac-20 beta) constructed was cotransfected into Spodoptera frugiperda Sf-9 cells with the baculovirus. After transfection, the recombinant virus was detected by the plaque assay with color selection. The expression of 3 alpha/beta(20 beta)-HSD cDNA was detected by Western blotting and enzyme assay. The expressed protein showed the same molecular weight and immunochemical cross-reaction to the native enzyme. Furthermore, it had 3-keto reductase activity of 3 alpha/beta(20 beta)-HSD for 5 alpha-dihydrotestosterone and 20-keto reductase activity for 17 alpha-hydroxyprogesterone in the presence of NADPH.

Cloning and primary structure of murine 11 beta-hydroxysteroid dehydrogenase/microsomal carbonyl reductase.

Screening of a mouse liver lambda gt 11 cDNA library with a rat liver 11 beta-hydroxysteroid dehydrogenase cDNA (11 beta-HSDr1A) and subsequent screening with an isolated mouse probe, resulted in the isolation and structure determination of a mouse cDNA encoding an amino acid sequence which is very similar to human and rat 11 beta-hydroxysteroid dehydrogenases (78% and 86% similar, respectively), and also to other known vertebrate 11 beta-hydroxysteroid dehydrogenase structures. Open-reading-frame analysis and the deduced amino acid sequence predict a protein with a molecular mass of 32.3 kDa which belongs to the superfamily of the short-chain dehydrogenase proteins. The amino acid sequence contains two potential glycosylation sites. These data are in agreement with information on the glycoprotein character of the native enzyme. This kind of post-translational modification seems to be a determining factor concerning the equilibrium of the catalyzed 11 beta-dehydrogenation/11-oxo reduction step [Obeid, J., Curnow, K. M., Aisenberg, J. & White, P.C. (1993) Mol. Endocrinol. 7, 154-160; Agarwal, A.K., Tusie-Luna, M.T., Monder, C. & White, P.C. (1990) Mol. Endocrinol. 4, 1827-1832]. After in vitro transcription/translation of the mouse cDNA, immunoprecipitation with anti-(microsomal carbonyl reductase) serum and N-terminal sequence analysis of the purified protein confirms the identity of microsomal 11 beta-hydroxysteroid dehydrogenase with the previously described, microsomal-bound xenobiotic carbonyl reductase [Maser, E. & Bannenberg, G. (1994) Biochem. Pharmacol. 47, 1805-1812], and points to an involvement of the 11 beta-HSD1A isoform in the reductive phase-I metabolism of xenobiotic compounds, besides its endocrinological functions. The alignment and comparison to other hydroxysteroid dehydrogenase forms of the same protein superfamily allows the identification of important residues in the 11 beta-HSD primary structure.

Cloning of glutaryl-CoA dehydrogenase cDNA, and expression of wild type and mutant enzymes in Escherichia coli.

We have cloned, sequenced, and expressed cDNAs encoding wild type human glutaryl-CoA dehydrogenase subunit, and have expressed a mutant enzyme found in a patient with glutaric acidemia type I. The mutant protein is expressed at the same level as the wild type in Escherichia coli, but has less than 1% of the activity of wild-type dehydrogenase. We also present evidence that the glutaryl-CoA dehydrogenase transcript is alternatively spliced in human fibroblasts and liver; the alternatively spliced mRNA, when expressed in E.coli, encodes a stable but inactive protein. Purified expressed human glutaryl-CoA dehydrogenase has kinetic constants similar to those of the previously purified porcine dehydrogenase. The primary translation product from in vitro transcribed glutaryl-CoA dehydrogenase mRNA is translocated into mitochondria and processed in the same manner as most other nuclear-encoded mitochondrial proteins. Human glutaryl-CoA dehydrogenase shows 53% sequence similarity to porcine medium chain acyl-CoA dehydrogenase, and these similarities were utilized to predict structure-function relationships in glutaryl-CoA dehydrogenase.

Isolation of glyceraldehyde 3-phosphate dehydrogenase (Gapdh) cDNA from the distal half of mouse chromosome 16: further indication of a link between Alzheimer's disease and glycolysis

The amyloid precursor protein (APP), which is localized on both human chromosome 21 and its murine counterpart, chromosome 16 and which is involved in the formation of deposits in Alzheimer's disease, could be shown to bind effectively to a glytolytic enzyme: rat glyceraldehyde 3-phosphate dehydrogenase (Gapdh). We report here the isolation of a cDNA of murine Gapdh from mouse chromosome 16 (MMU16) originating from microclones of the distal part of MMU16 and the use of homologous genomic DNA sequences to further screen a cDNA phage library. The cDNA was sequenced, confirmed by polymerase chain reaction following reverse transcriptase (RT-PCR) and the open reading frame was expressed in vitro. The possible localization of Gapdh on MMU16 - which may provide a mouse model for Down's syndrome and Alzheimer's disease - may lead to new insights into glycolysis and its role in the two syndromes.

'Liver-type' 11 beta-hydroxysteroid dehydrogenase cDNA encodes reductase but not dehydrogenase activity in intact mammalian COS-7 cells.

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) catalyses the metabolism of corticosterone to inert 11-dehydrocorticosterone, thus preventing glucocorticoid access to otherwise non-selective renal mineralocorticoid receptors (MRs), producing aldosterone selectivity in vivo. At least two isoforms of 11 beta-HSD exist. One isoform (11 beta-HSD1) has been purified from rat liver and an encoding cDNA cloned from a rat liver library. Transfection of rat 11 beta-HSD1 cDNA into amphibian cells with a mineralocorticoid phenotype encodes 11 beta-reductase activity (activation of inert 11-dehydrocorticosterone) suggesting that 11 beta-HSD1 does not have the necessary properties to protect renal MRs from exposure to glucocorticoids. This function is likely to reside in a second 11 beta-HSD isoform. 11 beta-HSD1 is co-localized with glucocorticoid receptors (GRs) and may modulate glucocorticoid access to this receptor type. To examine the predominant direction of 11 beta-HSD1 activity in intact mammalian cells, and the possible role of 11 beta-HSD in regulating glucocorticoid access to GRs, we transfected rat 11 beta-HSD1 cDNA into a mammalian kidney-derived cell system (COS-7) which has little endogenous 11 beta-HSD activity or mRNA expression. Homogenates of COS-7 cells transfected with increasing amounts of 11 beta-HSD cDNA exhibited a dose-related increase in 11 beta-dehydrogenase activity. In contrast, intact cells did not convert corticosterone to 11-dehydrocorticosterone over 24 h, but showed a clear dose-related 11 beta-reductase activity, apparent within 4 h of addition of 11-dehydrocorticosterone to the medium.(ABSTRACT TRUNCATED AT 250 WORDS)

CDNA cloning and chromosome mapping of human dihydropyrimidine dehydrogenase, an enzyme associated with 5-fluorouracil toxicity and congenital thymine uraciluria.

The pig and human dihydropyrimidine dehydrogenase (DPD) cDNAs were cloned and sequenced. The pig enzyme, expressed in Escherichia coli, catalyzed the reduction of uracil, thymine, and 5-fluorouracil with kinetics approximating those published for the enzyme purified from mammalian liver. DPD could be expressed in significant quantities only when uracil was added to the bacterial growth medium. The pig and human enzymes contained 1025 amino acids and calculated M(r) = 111,416 and 111,398, respectively. Conserved domains corresponding to a possible NADPH binding site and FAD binding site were found in the NH2-terminal half of the proteins and two motifs of putative [4Fe-4S] binding sites were found near to the carboxyl terminus of the enzyme. The latter corresponds to the labile COOH-terminal fragment previously shown to contain the iron sulfur centers. A sequence encompassing a peptide corresponding to the uracil binding site was found between the NADPH/FAD-containing NH2-terminal portion of the protein and the iron-sulfur binding sites near to the COOH terminus. Thus, the DPD appears to be derived from at least three distinct domains. The DPYD gene was localized to the centromeric region of human chromosome 1 between 1p22 and q21.

Molecular Cloning and Characterization of the Mouse Medium-Chain Acyl-CoA Dehydrogenase cDNA

Medium-chain acyl-CoA dehydrogenase (MCAD) is one of the three straight-chain length-specific dehydrogenases involved in the first step of fatty acid oxidation. Inherited defects of acyl-CoA dehydrogenases occur in humans, and MCAD deficiency is the most common, We have cloned the coding and 3′ untranslated sequence of mouse MCAD cDNA. The mouse MCAD cDNA coding region is 1263 bp long with a 3′ untranslated region of 576 bp and encodes a 421-amino acid precursor protein. Comparing the nucleotide and deduced amino acid sequences of the mouse MCAD cDNA to rat and human MCAD cDNAs reveals considerable similarity between species. Amino acid residues where substitutions result in human MCAD deficiency are conserved in the mouse. Amino acid residues involved in important enzymatic functions are also conserved.

The characterisation of the shikimate pathway enzyme dehydroquinase from Pisum sativum

Peptides accounting for 157 residues of the bifunctional shikimate pathway enzyme, dehydroquinase/shikimate dehydrogenase, of Pisum sativum were sequenced. Three of the peptides were homologous to regions in Escherichia coli dehydroquinase and two to E. coli shikimate dehydrogenase. The pea dehydroquinase activity was inhibited by treatment with dehydroquinate plus sodium borohydride, establishing it as a type I dehydroquinase. Synthetic oligonucleotides designed from the amino acid sequence were used as PCR primers to amplify fragments of P. sativum cDNA. DNA sequence analysis showed that these amplified products were derived from dehydroquinase/shikimate dehydrogenase CDNA. The complete amino acid sequence of the dehydroquinase domain has been defined; it is homologous to all other type I dehydroquinases and is N-terminal.

Molecular Cloning of Human Class IV Alcohol Dehydrogenase cDNA

A cDNA encoding a new type of alcohol dehydrogenase was cloned from a human stomach cDNA library. PCR amplification of 5′-stretch human stomach λgt11 library, using degenerate inosine-containing oligonucleotide probes compatible with peptide sequences of human sigma-ADH, resulted in a single product. Subsequently, internal non-degenerate primers were constructed according to the sequences occurring in the product. By PCR with combinations of these new primers and λgt11 forward and reverse primers, fragments of the cDNA containing its 5′ and 3′ ends were amplified. The full length cDNA sequence has 1125 nucleotides with a 72% similarity to those of human class I ADH. The polypeptide sequence, predicted from the cDNA, corresponds to 373 amino acids with a high degree of similarity (96%) to fragments of σ-ADH previously reported. Northern hybridization analysis with the specific probe for the mRNA of this protein showed that it is expressed in the human stomach but not in the liver. These data indicate that the cDNA we cloned is that of human class IV ADH.

Acetaldehyde exposure causes growth inhibition in a Chinese hamster ovary cell line that expresses alcohol dehydrogenase.

Chronic ethanol exposure causes many pathophysiological changes in cellular function due to ethanol itself and/or the effects of its metabolism (i.e., generation of acetaldehyde and redox equivalents). However, the role of each of these effects remains controversial. To address these questions, we have developed a cell line that expresses alcohol dehydrogenase. This cell line permits separate examination of the effects of ethanol and its metabolite acetaldehyde on cell function. An expression vector for the mouse liver alcohol dehydrogenase was constructed and transfected into Chinese hamster ovary cells. Cells expressing alcohol dehydrogenase were identified by screening with allyl alcohol, which is metabolized by alcohol dehydrogenase to the toxic aldehyde acrolein. A number of cell lines were identified that expressed alcohol dehydrogenase. A-10 cells were selected for further study because of their high sensitivity to allyl alcohol, suggesting a high level of alcohol dehydrogenase expression. These cells expressed a mRNA that hybridizes with the alcohol dehydrogenase cDNA and had an alcohol dehydrogenase activity comparable to murine liver. When cultures of these cells were exposed to ethanol, acetaldehyde was detected in both the medium and cells. The acetaldehyde concentration in the medium remained constant for at least 1 week in culture and was a function of the added ethanol concentration. Chronic exposure of A-10 cells to ethanol resulted in a dose-dependent reduction in the number of cells that accumulated over 7 days. Ethanol-treated cells remained viable, and growth inhibition was reversible. Growth inhibition was blocked by the alcohol dehydrogenase inhibitor 4-methylpyrazole, suggesting that acetaldehyde and not ethanol was responsible for growth inhibition in these cells.

Regulation of human dihydrodiol dehydrogenase by Michael acceptor xenobiotics.

A human oxidoreductase (H-37) that is overexpressed in ethacrynic acid-resistant HT29 colon cells (Ciaccio, P. J., Stuart, J.E., and Tew, K.D. (1993) Mol. Pharmacol. 43, 845-853) has been identified as a dihydrodiol dehydrogenase. Translated protein from a dihydrodiol dehydrogenase cDNA isolated from a library prepared from ethacrynic acid-resistant HT29 cell poly(A+) RNA was recognized by anti-H-37 IgG and was identical in molecular weight with H-37. The isolated cDNA was identical in both nucleotide and amino acid sequences with the recently cloned liver dihydrodiol dehydrogenase (Stolz, A., Hammond, L., Lou, H., Takikawa, H., Ronk, M., and Shively, J.E. (1993) J. Biol. Chem. 268, 10448-10457). Using this cDNA as probe, we have examined its induction by Michael acceptors. The steady state dihydrodiol dehydrogenase mRNA level in the ethacrynic acid-resistant line was increased 30-fold relative to that of wild-type cells. Twenty-four hour treatment of wild-type cells with ethacrynic acid or dimethyl maleate increased mRNA 10-fold and 5-fold, respectively. These changes are accompanied by both increased protein expression and increased NADP-dependent 1-acenaphthenol oxidative activity in cell cytosol. In gel shift assays, compared to wild type controls, increased binding of NAD(P)H quinone oxidoreductase human antioxidant response element (hARE) DNA to redox labile protein complexes present in treated and resistant cell nuclear extract was observed. Ethacrynic acid induced CAT activity 2-fold in Hepa1 cells stably transfected with NAD(P)H quinone oxidoreductase hARE-tk-CAT chimeric gene construct. Thus, dihydrodiol dehydrogenase protein is inducible by de novo synthesis from mRNA by structurally related monofunctional inducer Michael acceptors. Altered in vitro binding of nuclear protein to the hARE is indirect evidence for the involvement of an element similar to hARE in the regulation of dihydrodiol dehydrogenase by these agents.

Sequence of rat mitochondrial glycerol-3-phosphate dehydrogenase cDNA. Evidence for EF-hand calcium-binding domains.

The FAD-dependent, mitochondrial glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) is an essential component of the glycerol phosphate shuttle and is abundant in the pancreatic insulin cell, skeletal muscle, and brain. Although DNA clones for this enzyme and its homologues have been isolated from bacteria and yeast, it has never been cloned from a higher eukaryote. We have cloned and sequenced cDNAs encoding the rat mitochondrial glycerol-3-phosphate dehydrogenase. The longest cDNA (2337 base pairs) encodes a deduced protein of 727 amino acids that shows strong homology to the yeast and bacterial FAD-dependent glycerol phosphate dehydrogenases. The amino terminus of the purified mature protein was sequenced and shows identity with the deduced amino acid sequence beginning with residue 43. The 42 preceding amino acids are consistent with a mitochondrial leader peptide. A highly conserved FAD-binding domain and conserved regions possibly involved with glycerol phosphate binding are present. An unexpected finding was the homology of the deduced protein to calmodulin. Analysis of the deduced protein sequence shows a region near the carboxyl terminus containing two sequences homologous to "EF-hand" calcium-binding domains that are not present in the shorter yeast and bacterial homologues. The second of these domains appears to have features compatible with considerable affinity for calcium, whereas the first does not. The finding of a potential calcium-binding region is consistent with the known enhancement by calcium of the mammalian enzyme activity at low substrate concentrations and the lack of a requirement for calmodulin. This is the first report of EF-hands in a metabolic enzyme or in a mitochondrial protein.

Expression of 17β-hydroxysteroid dehydrogenase in the rat ovary during follicular development and luteinization induced with pregnant mare serum gonadotrophin and human chorionic gonadotrophin

Antibodies against human placental 17 beta-hydroxysteroid dehydrogenase (17-HSD) and 17-HSD cDNA were used to study the expression of the corresponding enzyme in the immature rat ovary during follicular development and luteinization, which were induced by treating the animals with pregnant mare serum gonadotrophin (PMSG) or with PMSG followed by human chorionic gonadotrophin (hCG). Immuno-blot analysis indicated that the M(r) of the 17-HSD expressed in rat granulosa cells was 35,000, as previously shown for the human placental enzyme. In immunohistochemical studies of untreated immature rat ovaries, only the granulosa cells from small antral follicles were stained. One day after PMSG treatment, strong expression of 17-HSD was observed in the granulosa cells of growing Graafian follicles. A marked decrease in enzyme expression was observed in preovulatory follicles on day 2 of PMSG treatment, starting from the basal layers of granulosa cells and progressing toward the luminal cells. No 17-HSD expression was detected in luteinized follicles or corpora lutea 22 h after hCG injection. The stroma and theca cells were negative for 17-HSD staining. In Northern hybridization analyses, two 17-HSD mRNAs were detected (1.4 and 1.7 kb). The strongest expression for both mRNAs was detected after 1 day of PMSG treatment, coinciding with maximal immunostaining of the enzyme protein. Down-regulation of 17-HSD observed by immunohistochemistry was reflected in a similar decrease in mRNA expression and the signals were almost undetectable 22 h after hCG injection. Our data suggest that 17-HSD expression in rat granulosa cells is up-regulated during follicular development and, thereafter, the enzyme expression is down-regulated during luteinization.

Gene amplification and dual point mutations of mouse IMP dehydrogenase associated with cellular resistance to mycophenolic acid

Mouse neuroblastoma cells (NB) selected for 10 000-fold increased resistance to mycophenolic acid (NB-Myco) showed a 200–500-fold increase in IMP dehydrogenase protein, and the enzyme (IMP: NAD + oxidoreductase, EC 1.1.1.205) also exhibited a 2400-fold increased k i for mycophenolic acid and reduced catalytic efficiency (Hodges, S.D., Fung, E., McKay, D.J., Renaux, B.S., and Snyder, F.F. (1989) J. Biol. Chem. 264, 18137–18141). The molecular basis of these changes is the subject of the present study. The nucleotide sequence of IMP dehydrogenase cDNA from NB-Myco cells revealed four nucleotide changes. One of these changes did not result in a codon change, and a second one corresponding to methionine-483 was present in the parental NB mouse line. The remaining two nucleotide substitutions and deduced residue changes are: the C to T transition at base 998 relative to initiation of translation, altering threonine-333 to isoleucine; and the C to A transversion at base 1052, altering serine-351 to tyrosine. Evidence was also obtained for IMP dehydrogenase having undergone gene amplification. IMP dehydrogenase mRNA levels were 500-fold increased in NB-Myco cells as compared to parental NB cells. DNA dot blot analysis showed a 25-fold increase in IMP dehydrogenase gene copy number and restriction enzyme analysis revealed similar gene structure for NB and NB-myco cells.

Molecular cloning of multiple cDNAs encoding human enzymes structurally related to 3α-hydroxysteroid dehydrogenase

Rat liver 3α-hydroxysteroid dehydrogenase cDNA was previously cloned by us. In this study, we used the rat cDNA as the probe to screen a human liver lambda gt11 cDNA library. A total of four different cDNAs were identified and sequenced. The sequence of one of the cDNAs is identical to that of the human chlordecone reductase cDNA except that our clone contains a much longer 5′-coding sequence than previously reported. The other three cDNAs display high degrees of sequence homology to those of both rat 3α-hydroxysteroid dehydrogenase and human chlordecone reductase. Because 3α-hydroxysteroid dehydrogenase and human chlordecone reductase belong to the aldo-keto reductase superfamily, we named these human clones HAKRa to HAKRd. Northern blot analysis showed that the liver expresses the highest levels of all four clones. Expression of all four clones was also detected in the brain, kidney, lung, and testis, whereas the placenta expressed only the messenger RNA for HAKRb. Genomic blot analysis using HAKRb as the probe detected multiple DNA fragments hybridized to the probe and a high degree of restriction fragment length polymorphism, suggesting the complexity of this supergene family.