Murine liver arylsulfatase B processing influenced by region on chromosome 17

SM/J liver arylsulfatase B has a more rapid electrophoretic mobility and occurs as a series of more acidic isozymes following electrofocusing in narrow pH gradients than the liver enzyme from C57BL/6J mice. The SM/J and C57BL/6J electrofocusing patterns were both converted to a single isozyme with similar isoelectric points by pretreatment with neuraminidase, suggesting that the SM/J and C57BL/6J isozymes differed with respect to their sialic acid content. Arylsulfatase B electrofocusing and thermostability phenotypes segregated independently among progeny of SM/J×C57BL/6J crosses, suggesting that the electrofocusing phenotypes were not determined by different alleles at As-1, the putative structural locus for arylsulfatase B. Comparison of the joint segregation of hepatic acid phosphatase electrophoretic patterns and liver arylsulfatase B electrofocusing profiles revealed that the electrofocusing profiles may be determined by a region on chromosome 17 near or identical to Apl. Kidney, brain, and spleen arylsulfatase B electrofocusing patterns did not appear to differ between SM/J and C57BL/6J mice.This research was supported in part by Biomedical Sciences Research Support Grant RR-07030, by NIGMS Grant 1-RO1GM27707-01, and by Grant 1–570 from the National Foundation/March of Dimes.     

Comparative biochemistry of mammalian arylsulfatase C and steroid sulfatase

1. Hepatic arylsulfatase C (ASC) and steroid sulfatase (SS) from six of eleven mammals (rat, dog, baboon, cow, goat, and sheep) coeluted from DEAE-Sephacel as a single anionic species. A minor cationic peak of ASC and SS activity was also recovered from solubilized microsomes derived from the domestic cat. Characterization of the cationic activities indicated they were most likely contributed by a protein structurally related to the anionic isozyme. Properties of ASC and SS activities occurring in these seven species were most consistent with the presence of both activities in the same enzyme. 2. Guinea-pig liver SS activity was partitioned between an alkylsulfatase (hydrolyzing dehydroepiandrosterone sulfate (DHEAS)) and an arylsulfatase (hydrolyzing both estrone sulfate (E1S) and 4-methylumbelliferyl sulfate (4MUS) at a common active site). These enzymes were physically separable by ion-exchange chromatography and possessed distinct immunological and chemical properties. 3. Porcine, squirrel, and human livers possessed a major isozyme of ASC that lacked both E1S- and DHEAS-sulfatase activities. The human hepatic ASC was separable from SS by electrophoresis and was partially resolved from SS by DEAE-Sephacel chromatography. The ASC isozyme lacking SS activity was heat-labile in all three species.     

A gene affecting liver activities of three glycosidases in the house mouse

A gene locus is described controlling liver activities in the house mouse of three glycosidases, i.e., -galactosidase, -glucuronidase, and N-acetyl--hexosaminidase. An allele conferring low activity is present in the inbred strain LIS/A, and an allele for high activity is present in A/Br Af mice. The three enzyme activities are correlated with each other. The possible linkage between this gene and the Bgs locus on chromosome 9 is discussed.     

Biochemical and immunological characterization of genetic variants of phosphoglucose isomerase from mouse

Two electrophoretic variants of phosphoglucose isomerase (PGI) were purified from whole body extracts of DBA/2J and C57BL/6J mice by a substrate-affinity elution from an 8-(6-aminohexyl) amino-ATP-Sepharose column followed by preparative isoelectric focusing. Both PGI variants were shown to be dimers of the same molecular weight, sedimentation coefficient, and K _m for fructose-6-phosphate. The isoelectric points were found to be 8.4 and 8.7 for variants from DBA/2J and C57BL/6J mice, respectively. Differential thermal stability was observed for the two variants in 0.1 m tris-HCl buffer, pH 8.0, at 54 C; the half-lives of the purified PGI from DBA/2J and C57BL/6J mice were shown to be 3.4 and 1.8 min, respectively, under those conditions. Similar differences were observed for the enzyme variants in the crude homogenates. Antisera against PGI from DBA/2J mice were raised in rabbits. The variants from DBA/2J and C57BL/6J mice showed no significant differences in their respective inactivation curves by the antisera. Results of amino acid composition analyses and peptide mappings of the two PGI variants indicate that the genetic variation of this enzyme might result from a single charged amino acid substitution.D. J. C. is a National Institutes of Health Visiting Fellow.     

Sox regulates transcription of the sea urchin arylsulfatase gene

A 50 bp region from -194 bp to -144 bp of the arylsulfatase gene (HpArs) of the sea urchin, Hemicentrotus pulcherrimus, is related to the temporally regulated expression of this gene. This region contains a Sox (Sry-related HMG box)-binding site, and the introduction of sequence mutations to this site significantly reduced the activity of the HpArs promoter, even in the presence of the C15 enhancer, which consists of HpOtx and CAAT motifs. A protein that binds to the Sox-binding site in the 50 bp region of the HpArs gene was detected in nuclear extracts of mesenchyme blastulae and a protein synthesized in vitro using SoxB1 cDNA of another sea urchin, Strongylocentrotus purpuratus, also bound to this Sox site. These results suggest that HpSox, which is maternally expressed and remains abundant by the pluteus stage, is clearly implicated in regulation of the HpArs gene. The presence of a negatively acting cis element in this 50 bp region has also been detected.     

East asian hemoglobin type (Hbb p) in wild populations of the house mouse in Israel

Electrophoretic studies of hundreds of individuals showed that all wild populations of the house mouse in Israel are polymorphic for alleles Hbb ^d and Hbb ^p of the hemoglobin locus. No mouse carrying Hbb ^s was found. This finding contradicts the notion that Hbb ^p is limited to East Asian house mice.     

Genetic control of heat sensitivity and activity level of murine arylsulfatase B

BALB/c male mice possess twofold higher kidney p-nitrocatechol-SO4 arylsulfatase B than do A/HeJ male mice; however, their liver arylsulfatase activities are comparable. Twentyfold-purified kidney arylsulfatases B from these two strains have similar Michaelis constants, electrophoretic mobilities, pH optima, and inhibitor profiles; however, the BALB/c enzyme is more heat stable than the A/HeJ enzyme. BALB/c, C3H/HeJ, DBA/2J, and SWR/J mice share an autosomal allele, As-1a, which apparently determines the heat-stable arylsulfatase B, while A/HeJ and C57BL/6J mice possess the As-1b allele, which determines the heat-sensitive enzyme. A second autosomal locus, Asr-1, determines liver arylsulfatase B activity. C57BL/6J mice carry the Asr-1a allele, which results in high liver activities, while C3H/HeJ mice are homozygous for the low-activity allele, Asr-1b. Male mice generally have 30-40% higher kidney activities than females; however, female kidney arylsulfatase activities rise and actually surpass those of males during late pregnancy and lactation.     

Comparative biochemistry of mammalian arylsulfatases A and B

1. Arylsulfatases A and B occurred as a major anionic and cationic isozyme, respectively, among eleven eutherian mammalian species. 2. Minor anionic arylsulfatase B isozymes were observed in rodents, dog, whale and pig, and were either monomeric (vole, Mr = 67 +/- 2 kDa), an apparent aggregate (dog, whale, pig; Mr = 192 +/- 10 kDa), or both (rat, mouse; monomeric Mr = 57 +/- 2 kDa; apparent dimeric Mr = 114 +/- 3 kDa). 3. Minor cationic arylsulfatase A isozymes were isolated from the deer, whale and pig. 4. Opossum arylsulfatases A and B were both anionic, had similar relative molecular weights, were not inhibited by silver, and were not precipitated by anti-murine arylsulfatase B nor anti-bovine arylsulfatase A IgG preparations.     

Linkage of loci affecting a murine liver protein and arylsulfatase B to chromosome 13

As-1 is the putative structural locus for murine arylsulfatase B, and Lth-1 determines the presence or absence of a 35 000 dalton acidic liver protein. As-1 and Lth-1 were found to be closely linked using recombinant inbred (RI) strains. Both loci were found to have been cotransferred with the pearl (pe) coat color mutation (chromosome 13) in the B6.C3H pe/pe congenic strain. The linkage relationships between pe, Lth-1, and As-1 were further defined in a backcross. On the basis of the RI data, the congenic strain result, and the backcross data, the following genetic distances were estimated: pe--As-1, 7.1 +/- 4.0 cM; As-1--Lth-1, 2.5 +/- 1.0 cM; and pe--Lth-1, less than 6.9 cM. As-1 and Lth-1 are the first biochemically defined loci to be added to the chromosome 13 linkage map.     

Enzyme variation and taxonomy: the estimation of sampling errors in measurements of interspecific genetic similarity

The uses of biochemical genetics as an aid to taxonomy are discussed briefly. A survey is made of published data to establish expected levels of genetic similarity or identity between conspecific populations, between congeneric species and between species of different genera. Existing measures of genetic similarity, identity or distance are discussed and some of the difficulties involved in their use in taxonomy are outlined. A new measure of genetic similarity is proposed specifically designed for use in making interspecific comparisons for taxonomic purposes. This measure offers considerable advantages in ease of calculation and also greatly simplifies the estimation of sampling errors. An empirical comparison is made, using published data, of the measures and confidence limits obtained by the proposed memod against published measures of genetic variation between species.     

Genetic variation in restriction patterns among mouse amylase gene complexes

The expression of pancreatic amylase in the mouse exhibits pronounced genetic variation. Congenic lines with various amylase complexes on a common C3H/As background have different numbers and forms of isoenzymes. The relative ratio of these isoenzymes may vary, as does the overall production of pancreatic amylase, which in some lines is three- to fourfold higher than in others. DNA from a number of lines was digested with endonucleases and hybridized to an amylase cDNA probe. The restriction patterns from inbred stocks and the corresponding congenic lines are identical, demonstrating that the majority of (if not all) amylase-like DNA sequences is found within the amylase complex. Congenic lines with specific amylase expression, for instance, in enzyme production, show different restriction patterns, whereas three lines with the same amylase phenotype have a uniform pattern. Most of the variation in amylase expression is represented among congenic lines derived from Danish mice. A comparison of such lines with others of remote geographic origin reveals that the restriction patterns of the Danish lines have by far the highest degree of resemblance. This observation seems to exclude major rearrangements within the amylase complex as the cause of the differences in enzyme expression, which instead are likely to be due to variation in regulatory elements associated with the active structural amylase genes in the complex.This work was supported by Grant 11-2290 from the Danish Natural Science Research Council.     

The control of IgM expression in a murine B cell lymphoma

The regulation of IgM expression was studied in clones derived from a murine B lymphocyte cell line, WEHI279.1. During normal B cell development IgM heavy chain synthesis increases concomitantly with heightened IgM secretion and reduced cell-surface IgM. However, in these subclones, the levels of membrane-bound and secreted IgM were regulated independently of one another. The amount of IgM secreted by the cells was tightly coupled to the amount of heavy chain synthesis, suggesting that the major control of secretion is pretranslational. Surface IgM exhibited a more complex regulation, with both pre- and posttranslational components. Variation in the expression of both forms of IgM occurred at high frequency. Although IgM expression follows a unidirectional pathway in nontransformed cells, the variability in these tumor cells was reversible and cellautonomous. High levels of phenotypic variability may be important in the ability of transformed cells to escape the immune response.     

Carbonic anhydrase is induced prior to arylsulfatase under the simultaneous deprivation of inorganic carbon and sulfate in Chlamydomonas reinhardtii (Volvocales, Chlorophyta)

The activity of periplasmic arylsulfatase (Ars), which catalyzes the cleavage of sulfate from aromatic sulfur compounds, was detected in cells acclimated to the sulfate-deficient conditions in a unicellular green alga Chlamydomonas reinhardtii Dangeard, but not in Chlorella, Scenedesmus, Dunaliella and Porphyridium. Upon the transfer of cells to sulfate-deficient autotrophic media under high-CO₂ conditions, the induction of Ars was observed only in the light, but not in the light with dichlorophenyldimethylurea (DCMU) nor in the dark. However, Ars was induced in the light with DCMU or in the dark when acetate was present as an organic carbon source, but not citrate. Under similar high-CO₂ conditions, high-CO₂ requiring mutants of cia-3 and cia-5, whose photosynthetic activities are greatly limited under low CO₂, showed much lower level of Ars activities than wild type cells. Under Iow-CO₂ conditions the induction of Ars was greatly suppressed even in wild type and no induction was observed in both mutants. These results suggest that the stimulation of photosynthetic or respiratory carbon metabolism are necessary for the induction of Ars. In contrast, the induction of periplasmic carbonic anhydrase (CA) which was synthesized de novo specifically under CO₂-limited conditions was strongly suppressed by the addition of organic carbon sources, such as acetate and citrate. When cells are subjected to CO₂-limitation and sulfate-deficiency simultaneously, the induction of CA was initiated immediately, while that of Ars was initiated following the completion of CA induction with an about 4-h lag. When the concentration of CO₂ was suddenly lowered during the induction of Ars, the induction of Ars ceased quickly, and the induction of CA was initiated instead. From these results the induction of CA was suggested to have priority over that of Ars under the dual stress of CO₂, and sulfate-deprivation.     

Evidence for the presence of two sympatric species of mice (genus Mus L.) in southern France based on biochemical genetics

Populations of mice established outdoors as well as indoors have been investigated at 24 loci using starch gel electrophoresis. Two reproductively isolated groups are recognized, one of which is referable to a house mouse subspecies, Mus musculus brevirostris, and the other to a different species, Mus spretus, contrary to the view of Schwarz and Schwarz that only one species of Mus is present in the Mediterranean Basin. The genetic distance between these two groups is larger than between any pair of investigated subspecies of M. musculus. M. m. brevirostris is biochemically almost indistinguishable from M. m. domesticus. On the other hand, M. spretus exhibits several allelic variants unknown or at most very infrequent in M. musculus, as for instance at the lactate dehydrogenase B-chain locus.This work was supported by research grants from the Centre National de la Recherche Scientifique (E.R.A. No. 261) and the Ecole Pratique des Hautes Etudes.     

Most human Alu and murine B1 repeats are unique

Alus and B1s are short interspersed repeat elements (SINEs) indirectly derived from the 7SL RNA gene. While most researchers recognize that there exists extensive variability between individual elements, the extent of this variability has never been systematically tested. We examined all Alu elements over 200 nucleotides and all B1 elements over 100 nucleotides in the human and mouse genomes, and analyzed the number of copies of each element at various stringencies from 22 nucleotides to full length. Over 98% of 923,277 Alus and 365,377 B1s examined were unique when queried at full length. When the criterion was reduced to half the length of the repeat, 97% of the Alus and 73% of the B1s were still found to be a single copy. All single and multi-copy sequences have been mapped and documented. Access to the data is possible using the AluPlus website http://www.ibr.hawaii.edu.     

A novel method for assessing gastritis in the murine model demonstrates genetically determined variation in response to Helicobacter felis infection

Background. In murine Helicobacter infection it has been demonstrated that the degree of gastric mucosal inflammation is determined by mouse strain. This is a valuable tool for investigating genetic, immunological or bacterial determinants for the outcome of human Helicobacter infection. This study aims to devise a robust method to facilitate these investigations. Materials and Methods. C57BL/6, BALB/c and (C57BL/6 × BALB/c) F1 mice were given 10⁸H. felis by gavage on days 1, 3 and 5 of the experiment Sections of the lesser and greater curve of stomach were examined at 12 weeks to assess active gastritis using a semi‐quantitative and a quantitative method by counting neutrophils in glands in four zones of the mucosa. Results. Semi‐quantitative scoring for active inflammation, based on the Sydney System, was inadequate with little discrimination between strains. Quantifying the level of active inflammation, counting the number of neutrophils in inflamed gastric glands and taking the total number of neutrophils in three inflamed pits within each zone (cardia, body, transitional zone and antrum) showed clear differences between the two parental strains for the degree of active gastritis and furthermore, using this system the phenotype of the (C57BL/6 × BALB/c) F1 was found to be between the two parental extremes. Conclusions. This novel method provides a numerical value for active inflammation in the stomach that is accurate, reproducible and discriminatory.