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.     

Genetics and ontogeny of alcohol dehydrogenase isozymes in the mouse: Evidence for a cis-acting regulator gene (Adt-1) controlling C2 isozyme expression in reproductive tissues and close linkage of Adh-3 and Adt-1 on chromsome 3

An electrophoretic variant previously reported for the stomach isozyme of alcohol dehydrogenase (ADH-C2) in inbred strains of Mus musculus (Holmes, 1977) has been used to localize the gene encoding this enzyme (Adh-3) on chromosome 3 near Va (varitint) (9.6 ± 3.6% recombinants). Genetic variation of ADH-C2 activity in male and female reproductive tissues among inbred strains and Harwell linkage testing stocks was also observed. Reproductive tissue ADH-C2 phenotypes were inherited in a normal Mendelian fashion among F₂ progeny of an F₁ (LII × C57BL/Go) × C57BL/Go backcross as though controlled by a single cis-acting regulator locus (designated Adt-1) with two alleles: Adt-1 ^a (presence of ADH-C2) and Adt-1 ^b (absence or low activity of ADH-C2). No recombinants were observed among 73 progeny or among 13 inbred strains and six Harwell linkage testing stocks of mice, indicating that Adh-3 and Adt-1 are closely linked or identical genes. A single recombinant phenotype was observed in Peru-Coppock mice, suggesting that they are separate genes. Ontogenetic analyses demonstrated that ADH-B₂ is present throughout development from late fetal stages in stomach, liver, and kidney; similar results were found for ADH-C2 in developing kidney and stomach extracts, whereas ADH-A2 exhibited high activity in liver extracts after 3 weeks of age in both sexes and in male kidney extracts after 6 weeks.     

The Genetics of α-Hydroxyacid Oxidase and Alcohol Dehydrogenase in the Mouse: Evidence for Multiple Gene Loci and Linkage between Hao-2 and Adh-3

Electrophoretic polymorphisms for stomach alcohol dehydrogenase (ADH-C2) and kidney L-alpha-hydroxyacid oxidase (HAOX-B4) have been identified in an Asian subspecies of mouse, Musmusculus castaneous. These variants are inherited in a normal Mendelian fashion with two alleles in each case showing codominant expression. The structural gene loci for those enzymes (Adh-3 and Hao-2, respectively) are apparently linked (17.6% recombinants) in this organism, whereas the multiple gene loci for HAOX, Hao-1 (encoding the A4 liver isozyme) and Hao-2, exhibited independent segregation and are unlinked (50% recombinants). Evidence is presented for 3 ADH loci: ADH-1, encoding liver ADH-A2 which exhibits high activity with ethanol (SELANDER, HUNT and YANG 1969; ADH-2, liver and stomach ADH-B2 using 2-hexene-1-ol as substrate; and Adh-3, stomach ADH-C2 using both benzyl alcohol and 2-hexene-1-ol as substrate.     

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.     

Esterase-23 (ES-23): Characterization of a new carboxylesterase isozyme (EC 3.1.1.1) of the house mouse,genetically linked to ES-2 on chromosome 8

Genetic variation of a carboxylesterase isozyme (EC 3.1.1.1) of the house mouse, designated ES-23, is described. ES-23 was found in kidney, liver, and intestine. The isozyme was resistant to inhibition by 10(-3) mol/liter eserine and was stained using alpha-naphthyl butyrate or 5-bromoindoxyl acetate as substrate. Five different phenotypes, ES-23A to ES-23E, could be distinguished by disc electrophoresis and by isoelectric focusing. ES-23 is controlled by a structural locus situated within the esterase gene cluster 2 on chromosome 8. An analysis of allele distribution among different strains suggested a separate structural locus for the isozyme, Es-23e, which is closely linked to the loci Es-2, Es-5, Es-7, and Es-11. Of the five phenotypes, only ES-23B was expressed in lung. This variation is apparently controlled by a cis-acting regulatory element, presumably a temporal locus, Es-23t, closely linked to the presumed structural locus Es-23e.     

Electrophoretic analyses of alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, sorbitol dehydrogenase and xanthine oxidase from mouse tissues.

1. Cellulose acetate zymograms of alcohol dehydrogenase (ADH), aldehyde dehydrogenase, sorbitol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase extracted from tissues of inbred mice were examined. 2. ADH isozymes were differentially distributed in mouse tissues: A2--liver, kidney, adrenals and intestine; B2--all tissues examined; C2--stomach, adrenals, epididymis, ovary, uterus, lung. 3. Two NAD+-specific aldehyde dehydrogenase isozymes were observed in liver and kidney and differentially distributed in other tissues. Alcohol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase were also stained when aldehyde dehydrogenase was being examined. 4. Two aldehyde oxidase isozymes exhibited highest activities in liver. 5. "Phenazine oxidase" was widely distributed in mouse tissues whereas xanthine oxidase exhibited highest activity in intestine and liver extracts. 6. Genetic variants for ADH-C2 established its identity with a second form of sorbitol dehydrogenase observed in stomach and other tissues. The major sorbitol dehydrogenase was found in high activity in liver, kidney, pancreas and male reproductive tissues.     

Androgen induction of alcohol dehydrogenase in mouse kidney. Studies with a cDNA probe confirmed by nucleotide sequence analysis

A cDNA clone for the beta-chain of human alcohol dehydrogenase (ADH) was used to isolate several cross-hybridizing clones from a mouse liver cDNA library. Clones pADHm9 and a portion of pADHm12 were sequenced. pADHm9 coded for a sequence of 151 C-terminal amino acids and some untranslated sequences from the 3' end of its corresponding mRNA. This clone was identified as an Adh-1 cDNA clone. Consistent with the known expression of Adh-1, this gene was expressed constitutively in liver, whereas the Adh-3 gene product was found only in stomach, lung and reproductive tissues. Furthermore, the translated region of the cDNA shared 91% amino acid sequence homology with rat liver ADH. [32P]pADHm9 was used as a hybridization probe to study the mechanism of androgen induction of kidney ADH activity. Induction of A/J female mice by androgen resulted in a dramatic increase in the steady-state level of Adh-1 mRNA content which correlated with the level of enzyme induction. The size of the mRNA obtained from control or induced kidney and liver tissues was indistinguishable by Northern analysis. [32P]pADHm9 was also used to probe restriction fragments of genomic DNA obtained from several inbred mouse strains. The hybridization patterns, considered with the genetic evidence, suggested that pADHm9 recognized sequences which may be present as only a single copy in the genome. No restriction fragment length polymorphisms were observed among the several inbred mouse strains examined.     

Biochemical genetics of aldehyde dehydrogenase isozymes in the mouse: Evidence for stomach- and testis-specific isozymes

Electrophoretic and activity variants have been observed for stomach and testis aldehyde dehydrogenases, respectively, among inbred strains of the house mouse (Mus musculus). Genetic evidence was obtained for two new loci encoding these isozymes (designated Ahd-4 and Ahd-6, respectively, for the stomach and testis isozymes) which segregated independently of a number of mouse gene markers, including Ahd-1 (encoding mitochondrial aldehyde dehydrogenase) on chromosome 4, ep (pale ears), a marker for chromosome 19, on which Ahd-2 (encoding liver cytosolic aldehyde dehydrogenase) has been previously localized, and Adh-3 (encoding the stomach-specific isozyme of alcohol dehydrogenase) on chromosome 3. Recombination studies have indicated, however, that Ahd-4 and Ahd-6 are distinct but closely linked loci on the mouse genome. An extensive survey of the distribution of Ahd-1, Ahd-2, Ahd-4, and Ahd-6 alleles among 56 strains of mice is reported. No variants have been observed, so far, for the microsomal (AHD-3) and mitochondrial/cytosolic (AHD-5) isozymes previously described. This study, in combination with previous investigations on mouse aldehyde dehydrogenases, provides evidence for six genetic loci for this enzyme.     

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 C57 BL/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 × 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.     

Liver aldehyde oxidase and xanthine oxidase genetics in the mouse

A 'null' activity variant for the major liver isozyme of aldehyde oxidase (AOX-1) in adult male mice and an electrophoretically distinct, high activity variant of the second liver isozyme (AOX-2) were used to examine the segregation of the genetic loci encoding these enzymes (Aox-1 and Aox-2 respectively) in breeding studies. A single recombinant between these loci was observed among the 147 backcross progeny examined, which confirms a previous report (Holmes, 1979) for close linkage and genetic distinctness of the two loci. An activity variant for mouse liver xanthine oxidase (XOX) is also reported which behaved as though controlled by codominant alleles at a single locus (designated Xox-1 ). Genetic analyses showed that the Xox-1 locus segregated independently of the multiple- A ox loci.     

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.     

α-Hydroxyacid Oxidase Genetics in the Mouse: Evidence for Two Genetic Loci and a Tetrameric Subunit Structure for the Liver Isozyme

We have examined a polymorphism for liver GOX in inbred strains of the mouse Mus musculus. Genetic studies demonstrated that the two phenotypes for this enzyme present in BALB/C and NZC parental strains segregated as though they were controlled by codominant alleles at a single autosomal locus (GOX) which mapped closely to the agouti locus in linkage group V. Kidney HAOX activity is invariant in these inbred strains and is encoded by a separate genetic locus designated HAOX. BALB/C x NZC F(1) hybrid mice exhibited three intermediate forms of liver GOX activity, in addition to the parental enzymes, which is consistent with a tetrameric subunit structure.     

Mapping of body weight loci on mouse Chromosome X

Inheritance of overweight in humans appears to be under polygenic control. Study on the mouse model may help to determine candidate regions in human genome for the search of overweight genes. Inbred mouse strains showed wide variation in body weight and can provide an experimental model for the study of inheritance of overweight. By genetic linkage analysis, we report the mapping of two loci, named Bw1 and Bw2 (body weight 1 and 2), on Chromosome (Chr) X that strongly affect adult body weight in two interspecific testcross male populations (HSB and ASB) of mice. In addition, another locus, named Bw3, is also mapped on Chr X in ASB populations. These loci account for up to 24% of the phenotypic variation in both populations. Considering the conserved synteny between mouse and human Chr X, these results provide candidate regions on Chr X that can be tested for linkage with overweight in humans.     

MuMTV antigen expression and mammary tumor incidence in non-inbred dd mice

The expression of mammary tumor virus (MTV) antigen in the milk and various organs of three non-inbred dd mouse stocks (ddO, ddN and ddY) was examined by the immunodiffusion (ID) and micro-immunodiffusion (micro-ID) tests. The rate of MTV antigen expression in the milk was 100% at the first lactation in ddO (6/6) and ddN mice (10/10), and 23% in ddY mice (3/13). Mammary tumor incidence was 13% (mean tumor age: 12.0 months), 32% (9.6 months) and 10% (11.5 months) in ddO, ddN and ddY mice, respectively, In F1 hybrids between MTV-free BALB/c females and dd males, a high level of MTV antigen was detected by the ID test in the milk of (BALB/c X ddO) F1, however, the levels in (BALB/c X ddN) F1 and (BALB/c X ddY) F1 mice were low at the first lactation and elevated with the advance of lactation number. Mammary tumor incidence had a trend to be higher and earlier in these F1 hybrids than in non-inbred dd stocks. The development of mammary tumors and detection of MTV antigen in F1 hybrids indicate the extrachromosomal transmission of MTV by male dd mice. The micro-ID test has shown that the mammary tumors, mammary glands, male genital organs except for the testis and the salivary gland expressed MTV antigen, with a high frequency of suggesting that secondary male genital organs may play an important role in MTV infection in mice.     

A gene (Bmn) controllingβ-mannosidase activity in the mouse is located in the distal part of chromosome 3

A gene (Bmn) with a major effect on -mannosidase activity in kidney and liver of the house mouse was revealed by assay with the synthetic substratep-nitrophenyl--d-mannoside. Activity is low in DBA/2J and CSB mice and high in C57BL/6J mice. By the use of the BXD series of recombinant inbred strains and by crosses between C57BL and CSB, it was possible to map the gene to the distal part of chromosome 3 by demonstration of linkage to a gene for cadmium resistance,cdm, as well as to theAdh-3 locus.This work was supported by Swedish Natural Science Research Council Project B-BU 2992-108.