Genetics and ontogeny of butyryl CoA dehydrogenase in the mouse and linkage of Bcd-1 with Dao-1

A zymogram method has been developed for fatty acyl CoA dehydrogenase and used to examine the electrophoretic properties of butyryl CoA dehydrogenase (BCD) from mouse tissues. A single form of BCD is present in extracts of liver, kidney, heart, and intestine. Ontogenetic, tissue distribution, and subcellular fractionation results are consistent with the mitochondrial origin previously reported for this enzyme. A genetic variant for BCD-1 was used to provide evidence for a locus determining the electrophoretic properties of this enzyme (designated Bcd-1), which is linked to Dao-1 (encoding d-amino acid oxidase).This research was funded in part by the Australian Research Grants Committee.     

Genetic regulation of alcohol dehydrogenase C2 in the mouse. Developmental consequences of the temporal locus (Adh-3t) and positioning of Adh-3 on chromosome 3

The tissue specificity of a proposed cis-acting temporal locus (Adh-3t), which regulates alcohol dehydrogenase C₂ (ADH-C₂) activity in mouse reproductive tissue extracts, has been examined in C5 7BL/6J, SM/J, F₁ (SM/J × C5 7BL/6J) mice as well as in progeny of an (F₁ [SM/J × C5 7BL/6J] × C5 7BL/6J) back-cross. Electrophoretic variants for ADH-C₂, previously used to localize the gene (Adh-3) encoding this enzyme on chromosome 3, enabled the relative parental contributions to ADH-C₂ phenotype in F¹ and backcross mouse tissues to be determined. These analyses demonstrated that (1) stomach, kidney, lung, adrenals, seminal vesicles, epididymis, uterus, and ovary ADH-C₂ is encoded by a single locus (Adh-3); Adh-3t is differentially active in various tissues, eg, lung exhibits no apparent activity whereas the temporal locus is fully active in seminal vesicles; (3) Adh-3t is probably differentically active in different cells of some tissues, eg, adrenals. Specific activity profiles of stomach and epididymal ADH-C₂ during the neonatal development of C5 7BL/6J, SM/J, and F₁ (SM/J × C5 7BL/6J) male mice supported the proposal for a cis-acting temporal locus for this enzyme. Genetic analyses examining segregation of Adh-3 and Adh-3t among backcross progeny suggested that these are distinct but closely linked loci, since one recombinant among 256 progeny was observed. Linkage data of Adh-3 with Va (varitint-waddler) and de (droopy ear) was also obtained, which suggested that Adh-3 is localized on chromosome 3 between Va and de.     

Dissociation-recombination of intergenic sunflower alcohol dehydrogenase isozymes and relative isozyme activities

Two unlinked genes, Adh ₁ and Adh ₂, control the production of alcohol dehydrogenase (ADH) in seeds of the annual sunflower (Helianthus annuus). Each gene is polymorphic, having F and S alleles. Starch gel electrophoretic zymograms of the four possible double homozygotes have three bands, representing two homodimers and an intermediately migrating intergenic isozyme. Zymograms of double heterozygotes consist of nine bands produced by ten isozymes: six intragenics and four intergenics, two of which are coincident. Results of dissociation-recombination (D-R) experiments are reported which demonstrate the subunit composition of the intergenic isozymes, thus supporting the relationships suggested by genetic studies. Densitometric tracings of the zymogram of a cleared gel and measurements of activities of homodimer isozymes eluted from gels following D-R of an intergenic isozyme showed that the Adh ₂ isozymes were more than twice as active as those of Adh ₁. Measurements of activities of crude extracts from the four possible double homozygous genotypes indicated that the seeds of the genotype Adh ₁ ^F /Adh ₁ ^F , Adh ₂ ^S /Adh ₂ ^S produced more activity than the other three. This genotype is the most common one found in wild and cultivated stocks. Isozymes eluted following electrophoresis of the same extracts had averages of 19%, 70%, and 11% of total activity contributed by the Adh ₁, Adh₂, and intergenic isozymes, respectively. A simple but efficient method of isozyme elution from starch gels is described which resulted in nearly full expected recovery (approximately 46%) of the ADH activity in the applied sample.Supported by Graduate School and BioMed grants and by NSF Grant GB35853.     

Aldehyde oxidase and alcohol dehydrogenase genetics in the mouse. New alleles for the Aox-2 and Adh-3 loci

The genetic variability of one of the liver isozymes of aldehyde oxidase (AOX-B₂ or AOX-2) and the stomach isozyme of alcohol dehydrogenase (ADH-C₂) has been examined among strains of mice. Evidence is presented for a fourth allele of Aox-2 and a third allele of Adh-3 . The hybrid allozyme pattern for mouse liver AOX was consistent with a dimeric subunit structure for this enzyme.     

Genetics,ontogeny, and testosterone inducibility of aldehyde oxidase isozymes in the mouse: Evidence for two genetic loci (Aox-1 and Aox-2) closely linked on chromosome 1

Null-activity and low-activity variants for the liver supernatant isozymes of aldehyde oxidase (designated AOX-1 and AOX-2) were observed in inbred strains and in Harwell linkage testing stocks of Mus musculus. The genetic loci determining the activity of these isozymes (designated Aox-1 and Aox-2, respectively) are closely linked on chromosome 1 near Id-1 (encoding the soluble isozyme of isocitrate dehydrogenase). Linkage data of Aox-1 with Id-1 and Dip-1 (encoding a kidney peptidase) demonstrated that this gene coincides with or is closely linked to Aox (Watson et al., 1972). Ontogenetic analyses demonstrated that liver AOX-1 appeared just before birth and increased in activity during postnatal development, whereas liver AOX-2 was observed only during postnatal development. Adult male livers exhibited higher AOX-1 and AOX-2 activities than adult female livers. Both isozymes were significantly reduced in activity by castration of adult males and increased following testosterone administration to castrated males and normal female mice.     

Genetics and ontogeny of aldehyde dehydrogenase isozymes in the mouse: Evidence for a locus controlling the inducibility of the liver microsomal isozyme

Variation in the inducibility of the liver microsomal isozyme of aldehyde dehydrogenase (designated AHD-Cy) by phenobarbital administration was observed among inbred strains and linkage testing stocks of Mus musculus. The phenotypes were inherited in a normal Mendelian fashion with two alleles showing codominance at a proposed regulatory locus (designated Ahd-3r). Strain variation was also observed for the induction of liver AHD-Cy by 17-β-oestradiol administration to ovarectimized female mice. Moreover, this enzyme was elevated in activity by the administration of high (nonphysiological) levels of progesterone. Development studies showed that the liver and kidney AHD-Cy isozyme exhibited low activities in late-stage fetal and neonatal mice and reached adult levels by approximately 6 weeks of age.     

Genetics of ocular NAD+-dependent alcohol dehydrogenase and aldehyde dehydrogenase in the mouse: Evidence for genetic identity with stomach isozymes and localization ofAhd-4 on chromosome 11 near trembler

Electrophoretic and activity variation of the stomach and ocular isozyme of aldehyde dehydrogenase (designated AHD-4) was observed between C57BL/6J and SWR/J inbred strains of mice. The phenotypes were inherited in a normal mendelian fashion, with two alleles at a single locus (Ahd-4) showing codominant expression. The alleles assorted independently of those atAdh-3 [encoding the stomach and ocular isozyme of alcohol dehydrogenase (ADH-C₂)] on chromosome 3. Three chromosome 11 markers, hemoglobin -chain (Hba), trembler (Tr), and rex (Re), were used in backcross analyses which established thatAhd-4 is closely linked to trembler. The distribution patterns for stomach and ocular AHD-4 phenotypes were examined among SWXL recombinant inbred mice, and those for stomach and ocular ADH-C₂ among BXD recombinant inbred strains. The data provided evidence for the genetic identity of stomach and ocular ADH-C₂ and of stomach and ocular AHD-4.This research was supported in part by the U.S. Department of Energy under Contract DE-ACO5-84OR214000 with Martin Marietta Energy Systems, Inc. (to R.A.P.).     

Genetics and ontogeny of aldehyde dehydrogenase isozymes in the mouse: Localization of Ahd-1 encoding the mitochondrial isozyme on chromosome 4

Electrophoretic variants for the mitochondrial isozyme of aldehyde dehydrogenase (AHD) have been observed in inbred strains and in Harwell linkage testing stocks of Mus musculus. F₁ (LVC×C57BL/Go) mice showed a codominant allele three-banded phenotype, which suggests a dimeric subunit structure (designated AHD-A₂). The anodal-migrating supernatant isozyme of AHD was electrophoretically invariant among the 23 inbred strains and stocks examined. The genetic locus encoding AHD-A₂ (suggested name Ahd-1) is localized on chromosome 4 and was mapped close to je (jerker) and Gpd-1 (encoding the liver and kidney isozyme of glucose-6-phosphate dehydrogenase). Ontogenetic analyses demonstrated that both AHD isozymes exhibited low activity in late fetal and early neonatal liver and kidney extracts, and reached adult levels within 3 weeks of birth.     

马肝醇脱氢酶基因的克隆及其在大肠杆菌中的表达

利用RT-PCR技术从马肝扩增HLADH-E和HLADH-S基因,通过基因工程方法构建表达质粒pLY115E和pLY115S,在大肠杆菌中表达,并利用Ni柱分离纯化。利用紫外检测辅酶NADH在340nm的吸光值,来考察表达产物转化环己醇的活性。试验结果证明马肝醇脱氢酶HLADH-E和HLADH-S基因均能在大肠杆菌中表达,并且可溶性表达产物都具有氧化环己醇的活性,为马肝醇脱氢酶的进一步研究开发奠定了基础。     

Genetical and biochemical comparisons of alcohol dehydrogenase isozymes from Anastrepha fraterculus and A. obliqua (Diptera: Tephritidae): Evidence for gene duplication

The electrophoretic patterns of the enzyme alcohol dehydrogenase (ADH) from Anastrepha fraterculus and A. obliqua were studied. Two loci were found to code for the enzyme in A. fraterculus, and three in A. obliqua. In both species, all isozymes were active in third-instar larvae. A cationic isozyme (Adh-1) was active mainly in the visceral fat body of both species. In A. fraterculus, the locus had an anionic polymorphic isozyme (Adh-3) that was detected in the parietal fat body. In addition to these two loci, a third locus for an anionic isozyme (Adh-2), which was active in the digestive tube of larvae, was present in A. obliqua and probably resulted from gene duplication. For both species, multiple forms of the isozymes are formed by binding of an NAD-carbonyl compound, as in Drosophila melanogaster. Both larvae and early pupae of A. obliqua had almost twice the specific ADH activity as A. fraterculus. The ethanol content of the host fruit infested with A. obliqua (red mombim) was also higher than that of the host fruit infested with A. fraterculus (guava).This research was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnologico (CNPq-PIG 40.2486/82).     

Alcohol dehydrogenase isozymes in grain sorghum (Sorghum bicolor): Evidence for a gene duplication

Two sets of alcohol dehydrogenase (ADH) bands are regularly observed in grain sorghum (Sorghum bicolor): set I is a permanent triplet; set II is variable, as either two or three bands. A faint set III is detected only when extracts from seeds subjected to anerobiosis are run in neutralpH gels. Dissociation-reassociation experiments reveal that the central band of the set I triplet is a heterodimer of the other two. Full-sib progeny analysis from selfed plants shows that the set II bands are doublets, with heterozygotes having only three apparent bands instead of four because of the similar mobilities of the fast-migrating isozyme specified by the slow allele and the slow isozyme specified by the fast allele. We propose a three-locus model as the best explanation of these patterns. Set I consists of the products of two loci and their intergenic heterodimer. Set III is specified by a third locus. Set II isozymes are the intergenic heterodimers of the two set I loci and the set III locus. This explanation is similar to that of Schwartz and Freeling for maize but suggests that the evolution ofSorghum includes a gene duplication of the homologue of theAdh-1 locus inZea. Supported by USDA Grant 59-2063-01522 to NCE and KWF.     

Heterologous expression of Zygosaccharomyces rouxii glycerol 3-phosphate dehydrogenase gene (ZrGPD1) and glycerol dehydrogenase gene (ZrGCY1) in Saccharomyces cerevisiae

We examined the effects of heterologous expression of the open reading frames (ORF) of two genes on salt tolerance and glycerol production in a Saccharomyces cerevisiae strain deficient in glycerol synthesis (gpd1Deltagpd2Delta). When the ORF of the Zygosaccharomyces rouxii glycerol 3-phosphate dehydrogenase gene (ZrGPD1) was expressed under the control of the GAL10 promoter, salt tolerance and glycerol production increased; when the ORF of the glycerol dehydrogenase gene (ZrGCY1) was expressed under the control of the GAL1 promoter, no such changes were observed. Zrgcy1p had a weak effect on glycerol production. These results suggest that Zrgpd1p is the primary enzyme involved in Z. rouxii glycerol production, following a mechanism similar to that of S. cerevisiae (Gpd1p). When the ORFs of the S. cerevisiae glycerol 3-phosphatase gene (GPP2) and ZrGPD1 were simultaneously expressed, glycerol production increased, compared with that in yeast expressing only ZrGPD1.     

Alcohol dehydrogenase isozymes in the mouse: Genetic regulation, allelic variation among inbred strains and sex differences of liver and kidney A2 isozyme activity

Genetic analysis of a proposed cis-acting temporal locus ( Adh-3t ), which regulates alcohol dehydrogenase C₂ (ADH-C₂) acitivity in mouse epididymis extracts, among F₁ (ddN × BALB/c) × ddN male backcross progeny provided evidence for genetic distinctness between the structural ( Adh-3 ) and temporal ( Adh-3t ) loci on chromosome 3. Genetic analysis also confirmed the close, linkage of Adh-1 (encoding liver and kidney ADH-A₂) and Adh-3 (encoding stomach ADH-C₂) to within 0.3 centimorgans on the mouse genome. Evidence is presented for a proposed closely linked cis-acting temporal locus (designated Adh-1t ) for the A₂ isozyme (encoded by Adh-1 ) controlling the activity of this enzyme in mouse kidney extracts, but having no apparent affect on liver and intestine ADH-A₂ activities. An extensive survey of the distribution of Adh-1, Adh-3 and Adh-3t alleles among 65 strains of mice is reported — with the exception of two Japanese strains (ddN and KF), linkage disequilibrium between Adh-3 and Adh-3t was observed. Sex differences in mouse liver and kidney ADH-A₂ activities were observed, with male/female ratios of approximately 0.6 and 3 respectively for these tissue extracts.     

Evidence for a possible regulatory gene (Suc-1) controlling sucrase expression in mouse intestine

Assays for sucrase carried out on intestinal sonicates prepared from 18 different strains of mice revealed a threefold variation in specific activity, the values for CBA/Ca mice being significantly less than for any other strain. Further comparison of the CBA/Ca versus the C57BL/6J mouse showed this deficiency, which became established 2–4 weeks after birth, to apply to isomaltase as well as sucrase but not to maltase or trehalase. Backcross experiments indicated that this deficiency in sucrase activity was inherited as a single codominantly expressed genetic factor. The ability of the CBA/Ca mouse to regulate sucrase activity in response to changes in diet was also reduced compared to that of the C57BL/6J mouse. No difference could be detected in the affinity of sucrase for its substrate or in the ability of heat to denature sucrase prepared from CBA/Ca and C57BL/6J mice. It is suggested that part of the regulatory region of the gene coding for sucrase-isomaltase is modified in the CBA/Ca mouse and that this locus should be given the notation Suc-1 for future reference.This work supported by an MRC project grant to M. W. Smith.     

Polymorphism of human liver alcohol dehydrogenase: Identification of ADH2 2-1 and ADH2 2-2 phenotypes in the Japanese by isoelectric focusing

Liver homogenate-supernatants from most Japanese exhibit an atypical pH optimum for ethanol oxidation at pH 8.8 instead of 10.5, the typical pH-activity optimum. It has been proposed that atypical livers contain alcohol dehydrogenase isozymes with ₂ subunits while typical livers contain isozymes with ₁ subunits, both produced by the ADH ₂ gene. Because it is difficult to differentiate the atypical ADH₂ 2-2 phenotype from the ADH₂ 2-1 phenotype by starch gel electrophoresis, an agarose isoelectric focusing procedure was developed that clearly separated the atypical Japanese livers into two groups, A1 and A2. The isozymes in A1 and A2 livers were purified. Type A1 livers contained a single isozyme with an atypical pH-rate profile; it was designated ₂₂. Three isozymes were isolated from A2 livers, two of which corresponded to ₁₁ and ₂₂. A third, absent from the typical and the atypical A1 livers, had an intermediate mobility; it was designated ₂₁. Type A1 livers are, therefore, the homozygous ADH₂ 2-2 phenotype, and type A2 livers, the heterozygous ADH₂ 2-1 phenotype. The ADH₂ 2-2 phenotype was found in 53% of 194 Japanese livers, and the ADH₂ 2-1 phenotype, in 31%. Accordingly, the frequency of ADH ₂ ² was 0.68.This study was supported by U.S. Public Health Service Grant AA 02342.     

ADH enzyme activity and Adh gene expression in Drosophila melanogaster lines differentially selected for increased alcohol tolerance

In Drosophila melanogaster, alcohol dehydrogenase (ADH) activity is essential for ethanol tolerance, but its role may not be restricted to alcohol metabolism alone. Here we describe ADH activity and Adh expression level upon selection for increased alcohol tolerance in different life-stages of D. melanogaster lines with two distinct Adh genotypes: Adh(FF) and Adh(SS). We demonstrate a positive within genotype response for increased alcohol tolerance. Life-stage dependent selection was observed in larvae only. A slight constitutive increase in adult ADH activity for all selection regimes and genotypes was observed, that was not paralleled by Adh expression. Larval Adh expression showed a constitutive increase, that was not reflected in ADH activity. Upon exposure to environmental ethanol, sex, selection regime life stage and genotype appear to have differential effects. Increased ADH activity accompanies increased ethanol tolerance in D. melanogaster but this increase is not paralleled by expression of the Adh gene.     

Aldehyde reductase isozymes in the mouse: Evidence for two new loci and localization of Ahr-3 on chromosome 7

Evidence is presented for two new forms of mouse liver and kidney aldehyde reductase activity (designated AHR-3 and AHR-4) resolved using cellulose acetate electrophoresis zymogram techniques and stained by glyceraldehyde and NADPH as substrate and coenzyme, respectively. Activity variants were observed for those isozymes among inbred strains of mice and used in a genetic analyses to support a proposal for two new genetic loci (Ahr-3 and Ah-4) which control the activity phenotype for these isozymes. Segregation analysis indicated that these loci are separately localized on the mouse genome, with Ahr-3 positioned on the distal end of chromosome 7. Liver AHR-2 (or hexonate dehydrogenase) exhibited no detectable phenotypic variation among the 44 inbred strains of mice examined. The AHR-3 and AHR-4 isozymes were readily distinguished from AHR-1 [or aldehyde reductase A₂, described previously by Duley and Holmes (Biochem. Genet. 20:1067, 1982)], hexonate dehydrogenase (AHR-2), and alcohol dehydrogenase A₂ in terms of their differential substrate, coenzyme, and inhibitor specificities.     

Phenotypic linkage between single-nucleotide polymorphisms of beta3-adrenergic receptor gene and NADH dehydrogenase subunit-2 gene,with special reference to eating behavior

The beta(3)-adrenergic receptor gene (BAR-3) allelic variant (Trp64Arg and Arg64Arg) is correlated with obesity or non-insulin-dependent diabetes mellitus. The mitochondrial NADH dehydrogenase subunit-2 gene (ND2) variant (Mt5178A) is associated with longevity or less susceptibility to adult-onset diseases. The frequencies of both the variants are high among the Japanese population. Cross-sectional analysis of these variants was conducted to determine if they correlated well with life-style-related phenotypes and nutrient intake. The body fat rate in the BAR-3 variant+ND2 variant group was higher than those rates in the BAR-3 normal+ND2 variant, BAR-3 normal+ND2 normal. The BAR-3 normal+ND2 variant group preferred much carbohydrate and less animal protein compared with other three groups. A combination of SNPs of the nuclear BAR-3 and the mitochondrial ND2 genes may affect eating behavior besides the biochemical and metabolic process of signal transduction and electron transfer system.