Segregation of genetic determinants for murine glucuroinidase synthesis and loss in CXB recombinant-inbred strains

A set of recombinant-inbred strains developed from mouse strains BALB/c and C57BL/6 includes two β-glucuronidase phenotypes that are not seen in either of the progenitor strains. These new recombinant phenotypes indicate that glucuronidase levels are regulated by genes additional to the Gur locus, which is closely linked to the glucuronidase structural gene (Gus) and is known to regulate the rate of glucuronidase synthesis. In this study, induced rates of glucuronidase synthesis were determined for these recombinant-inbred strains, and rate constants for enzyme loss were calculated. The rate of synthesis was found to segregate with the Gus gene in all of the strains, and only the determinants for rate of enzyme loss recombined to give new phenotypes. It was concluded that at least two genes affect the rate of enzyme loss, that these genes are not closely linked to each other or to the Gur-Gus region on chromosome 5, and that no major determinants of glucuronidase synthesis segregate independently of Gur.     

Effect of potassium deficiency on mouse kidney lysosomal enzymes.

Mice of inbred strains A/J, C57BL/6J and C57BL/6J beige were kept on a K+-deficient diet for up to 40 days to determine the magnitude and mechanism of changes in tissue lysosomal enzymes. From days 10 to 40 glucuronidase activity increased 3-fold in kidney of K+-deficient mice, but there was little effect on beta-galactosidase or acid phosphatase activity. Similar increases in kidney glucuronidase activity occurred in inbred strains known to have genetically altered control of the synthesis (A/J) and secretion (C57BL/6J beige) of glucuronidase in kidney proximal-tubule cells. Deprivation of K+ did not affect glucuronidase activity in liver, spleen, lung and brain, but there was a 2-3-FOld increase in glucuronidase activity in heart in the C57BL/6J and C57BL/6J beige strains. As shown by specific antibody titration, increased glucuronidase activity in kidney of K+-deficient mice was accompanied by accumulation of enzyme molecules. Likewise in kidney of deficient mice there was an increased rate of synthesis of glucuronidase as measured by incorporation of labelled leucine into immunoprecipitable glucuronidase. In kidney of K+-deficient mice the elevated glucuronidase activity was found in both collecting-tubule and interstitial cells of the medulla. It is probable therefore that a significant fraction of the increased kidney lysosomal synthesis and enzyme activity is due to infiltrating cells.     

Biochemical and genetic evidence for a macromolecular -glucuronidase complex in microsomal membranes

In the mouse β-glucuronidase is present in both microsomes and lysosomes and the enzyme at both sites is coded by the same structural gene. Electrophoresis on polyacrylamide gels showed that liver, kidney and lung from normal strains contained five enzyme forms designated L, M1, M2, M3 and M4 in order of decreasing mobility toward the anode. Band L is found primarily in lysosomes and is a tetramer of 260,000 molecular weight. Bands M1 to M4 are found exclusively in microsomes and range in molecular weight from 310,000 to 470,000. The increase in molecular weight is due to sequential addition of an accessory protein chain. When glucuronidase is highly induced in kidneys of female mice by injection of dihydrotestosterone, a sixth electrophoretic form of glucuronidase, designated X, appears. Form X appears early in induction, is localized in microsomes, and has a molecular weight (260,000) equal to that of the tetramer form L.Mice homozygous for the eg ° mutation, and thus deficient in microsomal glucuronidase, completely lack the microsomal forms M1 to M4. They do contain form X, and this increases after testosterone induction in kidney. The form X present in eg ° mice is indistinguishable from the form X seen in normal induced kidney.It appears that mice synthesize two different tetrameric forms of glucuronidase from the same structural gene. One, form L, is lysosomal; the other, form X, gives rise to microsomal enzyme forms M1 to M4 by the successive addition of up to four accessory protein chains. The eg ° mutant is blocked in the conversion of X to M1.     

Aryl Hydrocarbon Hydroxylase Induction by Benzo[a]anthracene: Regulatory Gene Localized to the Distal Portion of Mouse Chromosome 17

Aryl hydrocarbon (benzo[a]pyrene) hydroxylase inducibility by benzo[a]anthracene was studied in 29 somatic cell hybrid clones, developed by fusing mouse spleen or peritoneal cells from four different inbred strains with hypoxanthine phosphoribosyltransferase-deficient Chinese hamster E36 cells. Karyotype analysis plus 25 markers assigned to 16 autosomes and the X chromosome were examined. In 28 of the 29 clones, the presence or absence of inducibility is associated with the presence or absence, respectively, of mouse chromosome 17.—Liver microsomal aryl hydrocarbon hydroxylase induction by 3-methylcholanthrene or benzo[a]anthracene was assessed in appropriate backcrosses with the Mus musculus molossinus, M. m. castaneus, MOR/Cv, PL/J, SM/J and DBA/2J inbred strains and in 13 NX8 recombinant inbred lines. Twenty-seven biochemical genetic markers representing all but four autosomes were tested for possible linkage with the hydroxylase inducibility, and no linkage was found. The hepatic Ah receptor was quantitated in 26 BXD recombinant inbred lines; the Ah phenotype did not match exactly any of the more than 70 genes with established strain distribution patterns representing 12 autosomes and at least five unlinked markers.—It is concluded that a major gene controlling aryl hydrocarbon hydroxylase inducibility by benzo[a]anthracene is located on chromosome 17. Because there is no significant linkage with any of three biochemical markers in the upper third of the chromosome, we conclude that the inducibility gene is located in the distal 40% of mouse chromosome 17. Whether this trait represents the Ah locus, i.e., the gene encoding the cytosolic Ah receptor, will require further study.     

A structural gene (Hdc-s) for mouse kidney histidine decarboxylase

The concentration of mouse kidney histidine decarboxylase (HDC) is modulated by estrogen, testosterone, and thyroxine in a tissue-specific manner. Variation in HDC levels between strains of mice can be used to investigate the genetic regulation of (i) enzyme structure, (ii) tissue specific expression, and (iii) induction and repression by hormones. Variation in the structure of HDC between different inbred strains of mice affecting its K _m for the cofactor pyridoxal-5-phosphate (PLP) and its heat stability has been discovered. The alternative phenotypes are additively inherited in crosses and the heat stability difference is due to alleles of a single structural gene, Hdc-s, which segregate among the BXD and BXH recombinant inbred strains. The allele Hdc-s ^b determines the heat-stable phenotype (C57BL substrains), and the allele Hdc-s ^d the heat-labile phenotype (DBA/2 and C3H/He strains). The alleles of the structural gene cosegregate with alleles of a regulatory gene previously named Hdc (determining kidney enzyme concentration); there were no recombinants among 38 RI strains. Therefore the two loci are less than 0.685 cM apart and comprise part of the HDC gene complex, [Hdc], on chromosome 2 of the mouse.This work was supported in part by an SERC studentship to S.A.M. and an MRC project grant to G.B.     

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.     

Lysosomal Dysfunctions Associated with Mutations at Mouse Pigment Genes

Melanosomes and lysosomes share several structural and biosynthetic properties. Therefore, a large number of mouse pigment mutants were tested to determine whether genes affecting melanosome structure of function might also affect the lysosome. Among 31 mouse pigment mutants, six had 1.5- to 2.5-fold increased concentrations of kidney beta-glucuronidase. Three mutants, pale ear, pearl and pallid, had a generalized effect on lysosomal enzymes since there were coordinate increases in kidney beta-galactosidase and alpha-mannosidase. The effects of these three mutations are lysosome specific since rates of kidney protein synthesis and activities of three nonlysosomal kidney enzymes were normal. Also, the mutants are relatively tissue specific in that all had normal liver lysosomal enzyme concentrations.--A common dysfunction in all three mutants was a lowered rate of lysosomal enzyme secretion from kidney into urine. While normal C57BL/6J mice daily secreted 27 to 30% of total kidney beta-glucuronidase and beta-galactosidase, secretion of these two enzymes was coordinately depressed to 1 to 2%, 8 to 9% and 4 to 5% of total kidney enzyme in the pale-ear, pearl and pallid mutants, respectively. Although depressed lysosomal enzyme secretion is the major pigment mutant alteration, the higher lysomal enzyme concentrations in pearl and pallid may be partly due to an increase in lysosomal enzyme synthesis. In these mutants kidney glucuronidase synthetic rate was increased 1.4- to 1.5-fold.--These results suggest that there are several critical genes in mammals that control the biogenesis, processing and/or function of related classes of subcellular organelles. The mechanism of action of these genes is amenable to further analysis since they have been incorporated into congenic inbred strains of mice.     

The <Emphasis Type="Italic">C1473G</Emphasis> polymorphism in gene <Emphasis Type="Italic">tph2</Emphasis> is the main factor mediating the genetically defined variability of tryptophan hydroxylase-2 activity in the mouse brain

Brain neurotransmitter serotonin is involved in the regulation of many physiological functions and types of behavior. The key enzyme of serotonin synthesis in the brain is tryptophan hydroxylase-2 (TPH-2). Linkage between the C1473G polymorphism in gene tph2 causing the replacement of Pro⁴⁴⁷ by Arg⁴⁴⁷ in TPH-2 molecule and enzyme activity in the mouse brain of 10 inbred strains was found. Association of the polymorphism with the TPH-2 activity in the brain of F₂ hybrids between strains C57BL/6 and CC57BR was shown. The results indicate that the C1473G polymorphism in gene tph2 is the main factor determining the genetically defined variability of enzyme activity in the mouse brain.     

The Ah Phenotype. Survey of Forty-Eight Rat Strains and Twenty Inbred Mouse Strains

Forty-four inbred and four randombred rat strains and 20 inbred mouse strains were examined for their Ah phenotype by determining the induction of liver microsomal aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity (EC 1.14.14.1) by intraperitoneal treatment with either β-naphthoflavone or 3-methylcholanthrene. All 48 rat strains were found to be Ah-responsive. The maximally induced hydroxylase specific activities of the ALB/Pit, MNR/Pit, MR/Pit, SHR/Pit, and Sprague-Dawley strains were of the same order of magnitude as the basal hydroxylase specific activities of the ACI/Pit, F344/Pit, OKA/Pit, and MNR/N strains. Six of the 20 mouse strains were Ah-nonresponsive (i.e. lacking the normal induction response and presumably lacking detectable amounts of the Ah receptor). The basal hydroxylase specific activities of the BDL/N, NFS/N, STAR/N, and ST/JN mouse strains were more than twice as high as the maximally induced hydroxylase specific activity of the CBA/HT strain.——To date, 24 Ah-nonresponsive mouse strains have been identified, out of a total of 68 known to have been characterized. The reasons for not finding a single Ah-nonresponsive inbred rat strain—as compared with about one Ah-nonresponsive inbred mouse strain found for every three examined—remain unknown.     

Progressive induction of β-glucuronidase in individual kidney epithelial cells

The magnitude and kinetics of β-glucuronidase induction in mouse kidney are determined by a cis-acting regulatory gene, Gus-r, that is closely linked to the enzyme structural gene. The accumulation of β-glucuronidase mRNA during induction is much slower than the turnover time of the mRNA, suggesting progressive acquisition of mRNA synthesizing capacity during induction. Counts of the numbers of induced cells present at various times of induction in strains carrying three different alleles of Gus-r show that all potentially responsive cells respond immediately. The level of induction is progressive in individual cells and does not involve continued recruitment of new cells into the induced population. It appears that during induction each chromosome becomes progressively more active in directing the synthesis of β-glucuronidase.     

Genetic effects on male mouse kidney glucuronidase activity

Kidney β-glucuronidase activity in C57BL/Kl and DBA/2/Kl male mice differs about tenfold, C57 giving low and DBA high values. Another C57 subline, C57BL/6J, has slightly higher activity than C57BL/Kl. There is an association between the kidney glucuronidase activity and coat color determined by the buff locus, which indicates that part of the variation is due to differences at the Gur locus. The bf allele per se raises the activity of the enzyme. The backcross distributions give evidence that at least one more locus is involved.     

The genetics of aryl hydrocarbon hydroxylase induction in mice: A single gene difference between C57BL/6J and DBA/2J

Twenty-one inbred strains of mice were surveyed for inducibility of hepatic aryl hydrocarbon hydroxylase (AHH) activity by the carcinogen 3-methylcholanthrene (MC). In 11 strains given MC, AHH activity increased 1.3- to 5-fold (inducible), whereas ten strains responded with a less than 0.5-fold increase (noninducible). Neither the inducible nor the noninducible class was homogeneous, and in each considerable variation was found in both the basal activity of AHH and the response to MC. Strains DBA/2J and C57BL/6J were chosen to represent the noninducible and inducible classes, respectively. In the crosses (C57BL/6 × DBA/2)F₁ × DBA/2 and (C57BL/6 × DBA/2)F₂, inducibility segregated as a single autosomal dominant gene. The gene symbols Ahh ⁱ and Ahh ⁿ are proposed for the alleles present in C57BL/6J and DBA/2J, respectively. No genetic linkage was found between the Ahh locus and the following loci: b, d, Es-1, Es-3, Gpd-1, Hbb, Id-1, Pgm-1, and sex. Some implications of this work in the study of mammalian enzyme induction and chemically induced carcinogenesis are discussed. There is a positive correlation between AHH inducibility and the development of an inflammatory response to the topical application of the carcinogen 7,12-dimethylbenzanthracene.     

A regulatory locus,Hdc-e,determines the response of mouse kidney histidine decarboxylase to estrogen

Levels of histidine decarboxylase (HDC; EC 4.1.1.22) activity in female mouse kidney are modulated by estrogen (administered as implanted pellets). In some inbred strains HDC activity is induced by estrogen, while in others the enzyme is repressed. Immunoprecipitation with an anti-fetal rat HDC antiserum has shown that induction and repression of HDC levels are due to changes in enzyme concentration. Segregation analysis has identified a single additively inherited regulatory locus, Hdc-e, which determines the response to estrogen. The allele Hdc-eb (C57BL/10) determines induction, and the allele Hdc-ed (DBA/2) determines repression. Preliminary evidence indicates cosegregation of Hdc-e alleles with alleles of another regulatory locus, Hdc-c (determining kidney HDC concentration), and therefore putative linkage of Hdc-e with the HDC gene complex on chromosome 2. This is the first report of a mammalian regulatory gene controlling two opposite mechanisms, induction and repression in response to a single effector.     

Deficiency of a mouse kidney metalloendopeptidase activity: immunological demonstration of an altered gene product

Meprin, an 85,000 molecular weight metalloendopeptidase is a major component of the kidney brush border membrane in mice. Some inbred mouse strains exhibit low levels of meprin activity. These strains were characterized by little, if any, protein in brush border preparations corresponding to the native enzyme. However, material exhibiting partial identity to meprin was identified by Ouchterlony immunodiffusion. Immunoblots of brush border proteins confirmed that this immunoreactive material was present but of higher molecular weight than the native enzyme. The implication of these data is that the structural gene for meprin is expressed, albeit incorrectly, in the low-meprin strains.     

Evolution and Variation of Renin Genes in Mice

Inbred strains of mice carry Ren-1, a gene encoding the thermostable Renin-1 isozyme. Ren-1 is expressed at relatively low levels in mouse submandibular gland and kidney. Some strains also carry Ren-2, a gene encoding the thermolabile Renin-2 isozyme. Ren-2 is expressed at high levels in the mouse submandibular gland and at very low levels, if at all, in the kidney. Ren-1 and Ren-2 are closely linked on mouse chromosome 1, show extensive homology in coding and noncoding regions and provide a model for studying the regulation of gene expression. An investigation of renin genes and enzymatic activity in wild-derived mice identified several restriction site polymorphisms as well as putative variants in renin gene expression and protein structure. The number of renin genes carried by different subpopulations of wild-derived mice is consistent with the occurrence of a gene duplication event prior to the divergence of M. spretus (2.75–5.5 million yr ago). This conclusion is in agreement with a prior estimate based upon comparative sequence analysis of Ren-1 and Ren-2 from inbred laboratory mice.     

Hormonal regulation of ornithine aminotransferase biosynthesis in rat liver and kidney.

The relative rates of ornithine aminotransferase (OAT) synthesis in vivo were studied by pulse-labeling rats with [4,5-³H]leucine, isolating the mitochondrial enzyme protein by immunoprecipitation with a monospecific antibody, dissociating the immunoprecipitates on sodium dodecyl sulfate-acrylamide gels, and determining the radioactivity in OAT. After 4 days of treatment with triiodothyronine (T₃), both the enzyme activity level and the relative synthetic rate of OAT in rat kidney were elevated over twofold. The level of hepatic OAT activity was unaffected by this treatment. Thyroidectomy caused a 50% drop in the basal level of OAT activity and synthesis in kidney but not in liver. Although the basal levels of activity and synthesis of both renal and hepatic OAT were unaffected by adrenalectomy, the glucagon induction of the enzyme in liver was enhanced by about one-third and the T₃ induction in kidney was suppressed 50% by this operation. After 4 days of treatment with estrogen, both the enzyme activity level and the relative synthetic rate of OAT in male rat kidney were elevated nearly 10-fold. Hepatic OAT activity and synthesis were unaffected by this regimen. Thyroidectomy almost completely abolished the estrogen induction of OAT in kidney. OAT induction by estrogen could be restored by treating thyroidectomized rats with T₃. Simultaneous administration of T₃ plus estrogen to intact rats produced a multiple effect, resulting in a striking 20-fold induction of renal OAT. Although administration of either T₃ or estrogen causes an increase in the synthesis of immunoprecipitable OAT protein in rat kidney, each of these hormones may induce OAT by a different mechanism.     

Comparison of glucokinase in C3H/He and C58 mice that differ in their hepatic activity

Partially purified preparations of the hepatic glucokinase from C3H/He and C58 inbred mice have been used to explore the molecular basis for the observed twofold difference in activity between the strains. The single codominant gene that appears to regulate activity, the alleles of which are designated Gk^a and Gk^b, respectively, for the two strains, could represent a structural gene change. This now seems unlikely because the mouse enzyme, although showing small differences from rat glucokinase, appeared to be identical in the two strains with respect to thermal stability, electrophoretic mobility in agarose gels, and kinetic properties such as the apparent K _m values for MgATP^2– and glucose and the unique cooperative interaction with the latter substrate. The enzymes also reacted identically in a range of immunological tests (double-diffusion, immunoelectrophoresis, immune precipitation and immune inhibition assays) and ELISA immune inhibition assays indicated that the twofold difference in activity was due to a similar difference in antigenically active enzyme. Genetic control over the physiologically significant regulation of enzyme amount is therefore probable.This work has been supported in part by a grant from the British Diabetic Association and a Training Studentship to PAJ from the Medical Research Council (U.K.).     

Understanding the cause of false negative β‐d‐glucuronidase reactions in culture media containing fermentable carbohydrate

Aims:  To explain the basis for false negative β‐glucuronidase reactions seen with culture media containing lactose as a carbon and energy source. Methods and Results:  Escherichia coli strains were assessed for their reactions in culture media containing a β‐d ‐glucuronidase substrate either with or without lactose. An assay was developed to test for the expression of β‐d ‐glucuronidase at pH 5·0 and pH 7·2. Strains of E. coli that gave false negative glucuronidase reactions on media containing lactose generally expressed lower concentrations of the enzyme β‐d ‐glucuronidase than strains that gave positive results, although the difference was by no means consistent. Most strains that were negative on lactose‐containing media expressed virtually no β‐d ‐glucuronidase activity at pH 5·0. Examination of colonies on Membrane lactose glucuronide agar (MLGA) from lightly polluted water showed that c. 10% of the E. coli present failed to yield green colonies on MLGA. Conclusions:  E. coli that failed to produce green colonies on MLGA produced lower levels of β‐d ‐glucuronidase than did strains that formed green colonies, the difference being greater at pH 5·0 than pH 7·2. The false negative rate for E. coli 10% which is similar to that experienced in the study that originally described MLGA. Significance and Impact of the Study:  Strains of E. coli that fail to produce typical colonies on MLGA might produce lower concentrations of the enzyme β‐d ‐glucuronidase. Whilst the enzyme activity is sufficient to be detected at pH 7·2, fermentation of lactose significantly lowers the pH of the medium and can result in reduced enzyme activity and therefore lack of detection. The false negative rate of c. 10% would be difficult to detect in routine laboratories as it would represent 1% or less of yellow colonies being identified as E. coli (assuming E. coli accounts for 10% of the total coliform population in drinking water).     

Kinetics of beta-glucuronidase induction by androgen. Genetic variation in the first order rate constant

Measurements of enzyme activity, rates of protein synthesis, and mRNA activity suggest that the induction of beta-glucuronidase in mouse kidney in response to androgen is regulated at a pretranslational level. Following an initial lag period, the rate and extent of induction follow the rules of simple turnover kinetics and can be described in terms of a zero order rate constant for acquisition of mRNA activity (ka) and a first order rate constant for loss of activity (kb). Genetic variation in kb, described here for the first time, alters the half-time and extent of induction. Variation in kb is independent of previously described variation in ka and, unlike changes in ka, is not associated with change in the lag time. The DNA sequences determining kb, like those determining ka, are genetically linked to the structural gene for beta-glucuronidase. Following the removal of androgen, beta-glucuronidase activity, rate of synthesis, and mRNA activity all decline rapidly with half-lives of 1-2 days. Even in the most rapidly inducing strains, this is significantly faster than the half-time for induction determined by kb. Furthermore, genetic variation in kb does not affect the rate of de-induction. These facts suggest that kb may not describe the turnover of beta-glucuronidase mRNA, but rather the turnover of another step in the induction process.