Zinc deficiency in murine milk underlies expression of the lethal milk (lm) mutation.

The inability of nursing pups to survive on milk of mice homozygous for the recessive mutation, lethal milk (lm), is correlated with a reduction in zinc levels of both milk and pup carcass. Administration of zinc to pups nursing on lmlm dams reduces the observed mortality and morbidity. It is suggested that lm alters zinc transport from maternal blood to milk and that its study may provide useful information for understanding the rare human disease, acrodermatitis enteropathica.

Murine mucopolysaccharidosis type VII. Characterization of a mouse with beta-glucuronidase deficiency.

We have characterized a new mutant mouse that has virtually no beta-glucuronidase activity. This biochemical defect causes a murine lysosomal storage disease that has many interesting similarities to human mucopolysaccharidosis type VII (MPS VII; Sly syndrome; beta-glucuronidase deficiency). Genetic analysis showed that the mutation is inherited as an autosomal recessive that maps to the beta-glucuronidase gene complex, [Gus], on the distal end of chromosome 5. Although there is a greater than 200-fold reduction in the beta-glucuronidase mRNA concentration in mutant tissues, Southern blot analysis failed to detect any abnormalities in the structural gene, Gus-sb, or in 17 kb of 5' flanking and 4 kb of 3' flanking sequences. Surprisingly, a sensitive S1 nuclease assay indicated that the relative level of kidney gusmps mRNA responded normally to androgen induction by increasing approximately 11-fold. Analysis of this mutant mouse may offer valuable information on the pathogenesis of human MPS VII and provide a useful system in which to study bone marrow transplantation and gene transfer methods of therapy.

Androgen responsiveness of the murine beta-glucuronidase gene is associated with nuclease hypersensitivity, protein binding, and haplotype-specific sequence diversity within intron 9.

The tissue specificity and genetic variability of the murine beta-glucuronidase (GUS) response to androgen provide useful markers for identifying elements which underlie this responsiveness. While GUS is expressed constitutively in all examined cell types, kidney epithelial cells uniquely exhibit a manyfold yet slow rise in GUS mRNA and enzyme levels when stimulated by androgens. Three major phenotypes of this androgen response have been described among inbred strains of mice: (i) a strong response in strains of the Gusa haplotype, (ii) a reduced response in strains of the Gusb and Gush haplotypes, and (iii) no response, as observed in Gusor mice. These response variants define a cis-active element(s) which is tightly linked to the GUS structural gene. Nuclease hypersensitivity scans of kidney chromatin within and surrounding the structural gene revealed an androgen-inducible hypersensitive site in intron 9 of the gene in Gusa but not in Gusor mice. When a radiolabeled fragment of Gusa DNA containing this hypersensitive site was incubated with kidney nuclear extracts and then subjected to gel electrophoresis, two shifted bands were observed whose levels were dramatically higher in extracts of androgen-treated than in those of untreated Gusa mice. The shifted bands reflect binding of a kidney-specific factor(s) to a 57-bp region of complex dyad symmetry in Gusa and Gusor mice which is partially deleted in Gusb and Gush mice. This binding site is located approximately 130 bp downstream of a glucocorticoid response element sequence motif which is totally deleted in [Gus]or mice. Taken together, our results suggest that the androgen responsiveness of GUS in murine kidney epithelial cells is controlled by elements within the proximal end of intron 9 of the GUS structural gene.

DNA determinants of structural and regulatory variation within the murine beta-glucuronidase gene complex.

The murine beta-glucuronidase (GUS) gene complex, [Gus], encompasses the GUS structural element, Gus-s, and a set of regulatory elements which serve to modulate Gus-s expression. Three common GUS haplotypes representing virtually all inbred strains of laboratory mice have been compared with respect to GUS mRNA sequence. Results of such comparisons revealed sequence variations which target the location of one of the GUS regulatory elements to sequences within Gus-s and which account for known electrophoretic and heat stability differences among GUS allozymes of the three common GUS haplotypes.

Apparent trans control of murine beta-glucuronidase synthesis by a temporal genetic element.

A difference in the heat-inactivation kinetics between the beta-glucuronidases of C3HeB/FeJ and C57B1/6J mice was utilized to assess the mode of action of a temporal genetic element in controlling the expression of the beta-glucuronidase structural gene Gus. The heat-inactivation kinetics of liver and kidney beta-glucuronidase from F1 C3HeB/FeJ X C57B1/6J animals were intermediate with respect to the parental enzyme patterns, suggesting that equal concentrations of the two allelic products were present in beta-glucuronidase tetramers of F1 progeny. beta-glucuronidase heteropolymers assembled in vivo under conditions where equal concentrations of the two structural alleles of the enzyme were known to be present also exhibited intermediate heat-inactivation kinetics. These observations are consistent with a trans mode of action of a genetic element that controls the rate of murine beta-glucuronidase synthesis.

Two genetic elements regulate murine beta-glucuronidase synthesis following transcript accumulation.

Mutant alleles of two genetic regulatory elements, which underlie a three- to sixfold reduction in beta-glucuronidase (GUS) activity levels, distinguish mice of the H haplotype from those of the other two common GUS haplotypes, A and B. Both elements are tightly linked to the GUS structural gene over which they exert control. One (Gus-u) exerts a cis-active effect upon GUS activity levels in all tissues at all times while the other (Gus-t) regulates GUS activity in trans after the 12th postnatal day in certain tissues. While previous studies show that differences in the rate of GUS synthesis account for the combined effects of these two elements in liver of adult mice, we demonstrate the separate effects of each on GUS synthesis at times during early postnatal development when their individual expressions can be distinguished. Assessments of the relative levels of S1 nuclease protection of a radiolabeled GUS antisense RNA probe after hybridization with total liver RNA preparations from adult mice of A and H haplotypes reveal no differences. These results argue that Gus-u and Gus-t exert their control of GUS expression subsequent to the accumulation of processed GUS transcripts.

Androgen regulation of murine beta-glucuronidase expression: identification and characterization of a nonresponse variant.

One of the major features of beta-glucuronidase (GUS) expression in inbred strains of the house mouse, Mus musculus, is the responsiveness of this enzyme to androgen stimulation in tubule cells of the kidney. Both GUS-specific and nonspecific mutations have been described which define genes that serve to control this response. During examination of the expression of GUS in the interbreeding subspecies, Mus hortulanus, a new GUS haplotype was uncovered that is characterized, in part, by a lack of GUS response to androgen stimulation in an apparently responsive kidney. Blot hybridization analyses of kidney RNA with a radiolabeled murine GUS cDNA shows this lack of response to be reflected in GUS mRNA levels. The difference in heat stability of GUS activity between M. hortulanus and a responsive inbred strain, ICR/Ha, was utilized to assess the contribution of each parent to kidney levels of GUS in androgen-treated and -untreated F1 progeny of these strains. The results, together with preliminary genetic studies, suggest that the element controlling this responsiveness (or the lack thereof) is cis-active and tightly linked to the GUS structural gene on chromosome 5. It is not known whether this element is identical to another GUS-specific, cis-active element, Gus-r, which also controls the androgen response of GUS in mouse kidney.

Androgen responsiveness of mouse kidney beta-glucuronidase requires 5'-flanking and intragenic Gus-s sequences.

Genetics studies of natural variants of the androgen response of mouse beta-glucuronidase (GUS) reveal a cis-active element closely linked to the GUS structural gene (Gus-s) that is necessary for this kidney-specific response. Results of our previous studies suggested sequences within or near an androgen-inducible deoxyribonuclease I-hypersensitive site (DH site) located in the ninth intron of Gus-s are associated with the androgen response of GUS. Using transgenic mice, we now demonstrate that at least two regions of sequence within Gus-s are involved in regulating the androgen response of GUS. The first, located within 3.8 kb of Gus-s 5'-flanking sequence, directs the response and its tissue specificity, while the second, located within a 6.4-kb fragment of Gus-s extending from the third through the ninth intron of Gus-s, protects the androgen responsiveness of the transgene from repressive influences of the insertion site.

Isolation and expression in Escherichia coli of a cDNA clone encoding human beta-glucuronidase.

Mucopolysaccharidosis type VII is a lysosomal storage disease resulting from a deficiency of beta-glucuronidase (BG) activity. To facilitate the investigation of mutation in the disease and provide molecular diagnostic tools for affected families, we have isolated human BG cDNA clones. The SV40-transformed human fibroblast cDNA library of Okayama and Berg [Mol. Cell. Biol. 3 (1982) 280-289] was screened with a fragment of a murine BG cDNA clone (pGUS-1). The 17 human cDNA clones (pHUG) isolated were identical by restriction mapping, varying only in length. The pHUG clones show 80% DNA sequence homology with pGUS-1 in a 198-bp PvuII-SstI restriction fragment. Both pGUS-1 and the pHUG clones contained an open reading frame (ORF) throughout the sequenced region with a predicted amino acid sequence homology of 73%. Expression in Escherichia coli of a 1150-bp fragment of pHUG-1 subcloned in pUC9 resulted in an isopropyl-thio-beta-galactoside (IPTG)-inducible 35-kDal fusion protein which was specifically immunoprecipitated by goat anti-human BG immunoglobulin G (IgG). This evidence provides direct confirmation that the pHUG cDNA clones correspond to human BG.

Turnover of murine beta-glucuronidase. Comparison among liver, kidney, and spleen and between lysosomes and microsomes.

The turnover properties of murine beta-glucuronidase in several tissues and at two subcellular sites have been determined by monitoring the radioactivity present in immunoprecipitated enzyme at a number of time points following the in vivo administration of a single radiolabeled protein precursor (either L-[3,4(-3H)]leucine or or NaH14CO3). In all experiments a considerable period of time was required for the attainment of maximum specific radioactivity in glucuronidase. Similar labeling kinetics was found when [3H]leucine incorporation was monitored in immunoprecipitated murine liver delta-aminolevulinate dehydratase. Half-life estimates of 2 to 3 days were obtained for glucuronidases of liver, kidney, and spleen. In most strains of inbred mice, it is known that approximately 60% of total liver glucuronidase activity resides in lysosomes, while 40% is within the membranes of the endoplasmic reticulum (Ganschow, R. E. and Paigen, K. (1968) Genetics 59, 335-349). These two subcellular forms of glucuronidase turn over at similar rates. Furthermore, the bulk of glucuronidase in the endoplasmic reticulum does not serve as precursor to lysosomal glucuronidase.

Genetic variation of milk proteins in mice.

A survey for qualitative and quantitative variation in milk proteins from 58 inbred strains of mice revealed two electrophoretic variants. One is in a whey acidic protein of milk of YBR mice and the other is in a curd protein of the Asian house mouse, Mus musculus castaneus. The whey acidic protein variant is shown to be under the control of a single Mendelian autosomal gene with alleles expressed in a codominant manner. This gene is designated Wap, is not identical to Eg, is not X linked, and is either unlinked or loosely linked to the coat color genes a and b.

Complete sequence and organization of the murine beta-glucuronidase gene.

The murine beta-glucuronidase structural gene (Gus-s) has been isolated from a BALB/cJ sperm DNA bacteriophage library and its nucleotide sequence established. The gene is organized into 12 exons comprising 17.5% of the 14,009 base pair (bp) region spanning the interval between transcription initiation and the putative site of polyadenylation. A TATA box sequence, embedded within a GC-rich region, is found 28 bp upstream from the transcription initiation site. Eleven members of the B1 family and eight members of the B2 family of murine repetitive elements were identified within Gus-s and 2440 bp of flanking sequence. Other novel sequences found within Gus-s, including a (AC)19 homocopolymer tract within intron 3 and a 23 base pair complex direct repeat within intron 9, are presented and discussed.

The complete nucleotide sequence of murine beta-glucuronidase mRNA and its deduced polypeptide.

The complete nucleotide sequence of murine beta-glucuronidase (GUS) mRNA has been compiled from three overlapping cloned cDNAs and a single GUS-specific genomic clone. The sequence is composed of 2455 nucleotides, exclusive of the poly(A) tail. The 5' and 3' untranslated regions contain 12 and 499 bases, respectively, with the open reading frame encoding a polypeptide of 648 amino acids (74.2 kDa), including a 22 amino acid signal sequence. The nucleotide and deduced amino acid sequences of murine GUS are compared to those published for rat and human GUS and the results are presented. Murine GUS also shares amino acid sequence identity with Escherichia coli GUS and beta-galactosidase. The complete sequences of murine GUS mRNA and its deduced polypeptide provide a basis from which to study the mechanisms responsible for the well-characterized variation in GUS expression among inbred mouse strains.

Genetic control of levels of murine kidney glucuronidase mRNA in response to androgen.

A cis-acting genetic element, designated Gus-r, regulates the androgen-induced rates of murine glucuronidase (EC 3.2.1.31) synthesis in kidney tubule cells and is tightly linked to the glucuronidase structural gene, Gus-s. To investigate the molecular mechanism underlying this regulation, we have cloned a glucuronidase-specific cDNA sequence in plasmid pBR322. This cloned DNA has been utilized as a probe in blot hybridization analyses to determine whether the control of androgen responsiveness of kidney glucuronidase synthesis by Gus-r is exerted over the level or the translatability of glucuronidase mRNA. Three important observations emerged from these studies: (i) glucuronidase mRNA exists as a single size class of approximately 2,800 nucleotides; (ii) androgen stimulation of glucuronidase synthesis is directly related to the level of glucuronidase mRNA; and (iii) strain differences in levels of kidney glucuronidase mRNA accumulated in response to androgen are controlled by alleles of Gus-r. Thus, Gus-r regulates the androgen responsiveness of glucuronidase synthesis by controlling the amount of glucuronidase mRNA available for translation and is a cis-acting genetic element that regulates the hormonal responsiveness of a specific mRNA.

The molecular genetics of mammalian glucuronidase.

The genetic factors known to be involved in the final realization of beta-glucuronidase activity in mice are considered from the standpoint of structural genes determining the catalytic activity of enzyme molecules as well as the recognition features of enzyme molecules that identify them for subsequent processing by the cell; processing genes determining the cellular apparatus involved with the conjugation, intracellular localization and eventual degradation of enzyme molecules; regulatory genes determining rates of enzyme synthesis, especially in response to physiological signals such as hormones; and temporal genes determining the developmental programs for expression of these classes during growth and differentiation. The properties of genetic variants of beta-glucuronidase falling into each of these classes are described. When those results are considered in concert with the properties of genetic variants known for other mammalian enzymes several generalizations emerge. Structural genes of enzymes are not usually linked to the processing genes determining the post-assembly events in the life of that enzyme. In contrast, all of the regulatory and temporal gene sites so far identified are in close proximity to the structural genes they modulate. Regulatory and temporal sites appear to act in a cis fashion to control the amount of enzyme synthesized from the adjacent structural allele on the same chromosome.

Zinc and copper in milk and tissues of nursing lethal milk mutant mice.

Zinc concentration was lower in liver of suckling 1-d-old lethal milk (lm/lm) mutant mice than in wild-type pups, in accordance with the hypothesis of milk-induced zinc deficiency previously proposed to underlie this mutation. Despite the initial deficiency, by 3 d of age suckling lm/lm pups exhibited higher levels of hepatic zinc than did lm/lm-nursed wild-type pups. Intestinal zinc and copper concentrations were normal in 1-d-old lm/lm pups, but by 3 d of age were also higher in lm/lm pups than in wild-type pups foster-nursed on lm/lm dams. Contrary to a previous report, we found that zinc concentration in milk of lm/lm dams was not significantly different from those of controls, between 4-20 d postpartum. Mutant milk showed normal zinc distribution as determined by gel-filtration chromatography or by DEAE-cellulose chromatography of zinc-binding ligands derived from EDTA-dissociated micelles, normal copper levels, normal amounts of citrate, a zinc (II) and copper (II)-binding ligand and normal amounts of glutamate, a proposed copper (II)-binding ligand. Total mammary glands and mammary gland cytosols from lm/lm mice exhibited normal zinc concentrations. Copper levels, however, were higher in lm/lm mammary gland cytosols than in controls. These results suggest that an increased uptake and/or retention of zinc and copper in the tissues studied may underlie the signs of zinc deficiency seen in lethal milk mutant mice.

Characterization of a murine cDNA encoding a member of the carboxylesterase multigene family.

We have characterized a mRNA sequence containing the entire coding region of a mouse carboxylesterase (EC 3.1.1.1). pEs-N, an 1840-bp composite of five overlapping cDNA clones, contains an open reading frame of 554 amino acids that display a high degree of similarity with rat and rabbit carboxylesterases. Genetic mapping studies place this carboxylesterase in cluster 1 of the esterase region on chromosome 8. Results of blot hybridization analysis of genomic DNA probed with a pEs-N cDNA under both low and high stringency conditions suggest membership in a carboxylesterase multigene family, as would be expected for a nonspecific carboxylesterase. A message size of 1850-1900 nucleotides was revealed by RNA blot hybridization analysis. S1 nuclease protection analyses with a probe representing a segment of pEs-N detected message in liver, kidney, and lung, but not in spleen, brain, testes, and submandibular gland, with higher levels in female than in male kidney. Additional S1 nuclease-protected mRNA species were found, suggesting the expression of distinct members of a multigene family. In vitro translation of a full-size transcript of pEs-N resulted in a product of 51.5 kDa. Upon the addition of microsomes, this product was processed into a protein of 60.4 kDa, which is within the size range of monomeric units of mouse carboxylesterases.