The accumulation and metabolism of glycosphingolipids in primary kidney cell cultures from beige mice

In the normal C57BL/6J male mouse a specific subset of the kidney glycosphingolipids which is associated with multilamellar bodies of lysosomal origin and represents about 10% of the total kidney glycolipids, is excreted into the urine each day. This excretion is blocked and glycosphingolipids accumulate in the kidney of bg ^J/bg^J mutants of this strain. To examine this process in vitro, glycosphingolipid metabolism and excretion were studied in beige mouse kidney cell cultures. Primary kidney cell cultures from male C57BL/6J control and bg ^J/bg ^J beige mutants were grown in D-valine medium and glycosphingolipids labeled with [³H]palmitate. As we have shown previously, the giant lysosomes of altered morphology were maintained in cultures of the beige kidney cells. Beige-J and control cells synthesized the same types of glycosphingolipids, but the mutant cells had quantitatively higher levels of these compounds than control cells, as determined by high performance liquid chromatography. Beige-J cells incorporated more [³H]palmitate into glycospingholipids than control cells on a cpm/mg protein basis and the specific activity (cpm/pmole glycosphingolipid) was lower in beige cells. Medium from beige-J cells accumulated more glycosphingolipids than that from control cells in a 24 h period. The glycosphingolipids released into the medium as determined by HPLC were primarily non-lysosomal types and both control and mutant cells retained the glycosphingolipids associated with lysosomal multilamellar bodies excreted in vivo. The elevated levels of lysosomal glycosphingolipids and the dysmorphic lysosomes in primary cultures of beige cells, then, are not caused by a mutant block in secretion of lysosomes. (Mol Cell Biochem 118: 61–66, 1992)     

Defective lysosomal enzyme secretion in kidneys of Chediak-Higashi (beige) mice

The beige mouse is an animal model for the human Chediak-Higashi syndrome, a disease characterized by giant lysosomes in most cell types. In mice, treatment with androgenic hormones causes a 20-50-fold elevation in at least one kidney lysosomal enzyme, beta-glucuronidase. Beige mice treated with androgen had significantly higher kidney beta-glucuronidase, beta-galactosidase, and N-acetyl-beta-D-glucosaminidase (hexosaminidase) levels than normal mice. Other androgen-inducible enzymes and enzyme markers for the cytosol, mitochondria, and peroxisomes were not increased in kidney of beige mice. No significant lysosomal enzyme elevation was observed in five other organs of beige mice with or without androgen treatment, nor in kidneys of beige females not treated with androgen. Histochemical staining for glucuronidase together with subcellular fractionation showed that the higher glucuronidase content of beige mouse kidney is caused by a striking accumulation of giant glucuronidase-containing lysosomes in tubule cells near the corticomedullary boundary. In normal mice lysosomal enzymes are coordinately released into the lumen of the kidney tubules and appreciable amounts of lysosomal enzymes are present in the urine. Levels of urinary lysosomal enzymes are much lower in beige mice than in normal mice. It appears that lysosomes may accumulate in beige mice because of defective exocytosis resulting either from decreased intracellular motility of lysosomes or from their improper fusion with the plasma membrane. A similar defect could account for characteristics of the Chediak-Higashi syndrome.     

UDP-galactose:globoside galactosyltransferase in murine kidney

There are increased levels of stage-specific embryonic antigens-3 and -1 (SSEA-3 and SSEA-1) globo-series glycolipids in male versus female DBA/2 and C57BL/6 kidneys, respectively. To determine what enzymatic steps may be responsible for these differences, the activity and properties of UDP-galactose:globoside galactosyltransferase were studied in male and female mouse kidney microsomes. This enzyme participates in the biosynthesis of galactosylgloboside, SSEA-3 glycolipid; the reaction product was identified by high performance thin-layer chromatography (HPTLC) immunostaining. In C57BL/6 mice, the specific activity of the enzyme, in the presence of CHAPS, was 2-fold greater in the male than that in the female. Optimum pH for the enzyme from both sexes was about 5.6, and Mn2+ was essential for maximal activity. Fifty percent of the male and female enzyme activity was lost after preincubating the microsomes for 1 min at 55 degrees C; thereafter, the enzyme from female microsomes had a slower rate of denaturation. The Km for globoside in presence of sodium cholate for both male and female was 0.035 mM, but it was approximately 2-fold greater for the female in presence of CHAPS. The enzyme in male and female microsomes was differentially activated by CHAPS and cholate. The results suggest the presence of an enzyme modulator in these membranes. In DBA/2 mice, the enzyme activity was about 2-fold greater in males than that in the female. The specific activity of the enzyme in the two strains was of a similar magnitude.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular distribution of lysosomal enzymes in the mouse pigment mutants pale ear and pallid

Summary The size distribution of lysosomes was determined in kidney proximal tubule cells of two mouse pigment mutants, pale ear and pallid, which have an increase in kidney lysosomal enzyme content caused by a decreased rate of secretion of lysosomal enzymes into urine. Both mutations have larger lysosomes when compared with normal mice. However, neither mutant contains the giant lysosomes (up to 11 micron diameter) common to the well-characterized beige mutant, which has a kidney secretory defect similar to the pale ear and pallid mutants. Subcellular distribution studies, performed by the osmotic shock technique, likewise suggested differences among the pigment mutants. A very high content of soluble enzyme, indicative of lysosomal fragility during homogenization, was found in extracts from the beige mutation. By comparison, the percent of soluble enzyme became progressively lower in extracts of the pallid and pale ear mutants and was lowest in extracts from normal mice. All 3 pigment mutants had normal concentrations of osmotically resistant membrane-bound lysosomal enzymes. This indicates that the excess, non-secreted, lysosomal enzyme in all three pigment mutants likely is present in classical lysosomal organelles rather than in other non-lysosomal subcellular membrane fractions. The results also illustrate that mammalian mutants which exhibit decreased lysosomal secretory rates can have strikingly different effects on morphology of lysosomes.Supported in Part by National Science Foundation Grant PCM 77-24804. E. K. N. was supported in part by United States Public Health Service Grant GM 007093-03.     

Biochemical and morphological characterization of primary kidney cell cultures from beige mutant mice

Summary Primary kidney cultures from adult beige-J (bg ^J/ bg ^J) mice were selected for epithelial cell growth using D-valine medium. After 2 weeks of attachment and proliferation in vitro, the cells form a confluent or nearly confluent monolayer that retains several phenotypic characteristics of the beige-J mutant. These include large, multilamellar inclusion bodies that are apparently dysmorphic lysosomes, and higher concentrations of neutral glycosphingolipids and dolichols than control cells. -Glucuronidase activity, used as a lysosomal enzyme marker, is not elevated in beige-J-cultured kidney cells compared with controls, as it is in the intact kidney. The high levels of -glucuronidase activity in both control and mutant cells may mask expression of this difference in vitro. The action of the beige-J mutation in kidney cells is thought to be due to a block in exocytosis that results in the accumulation of abnormal lysosomes and their components. The maintenance of the beige phenotype in vitro indicates that the mutation is not suppressed in primary kidney cell cultures. The expression of the beige phenotype in vitro should be useful for studies concerning the primary lesion of this mutation.     

Lipid composition of lysosomal multilamellar bodies of male mouse urine

Previous studies from our laboratory have shown that male C57BL/6J mice excrete into the urine multilamellar lysosomal bodies that contain specific neutral glycosphingolipids. These mice excrete approximately 20-30% of their kidney glycolipids each day. The significance and function of this secretion of multilamellar lysosomal organelles is unknown. To characterize these excreted bodies further, we report here their neutral lipid and phospholipid composition. The bodies were collected by differential centrifugation, extracted with chloroform-methanol, and lipids were fractionated into neutral lipids, glycolipids, and phospholipids. The neutral lipids consisted primarily of cholesterol, dolichol, and ubiquinone. The phospholipid fraction consisted primarily of a single molecular species of phosphatidylcholine. This lipid which comprises more than 90% of the total phospholipids was found to contain 16:0 ether and C22:6 n-3 fatty acid as determined by gas-liquid chromatography-mass spectrometry. The glycosphingolipids as reported previously consisted primarily of galabiosylceramides and globotriaosylceramides. This membrane lipid composition is different from any previously reported cellular organelle.     

Altered secretion and accumulation of kidney glycosphingolipids by mouse pigmentation mutants with lysosomal dysfunctions

The kidney and urine glycosphingolipids of five pigmentation mutants which are known to have altered secretion of kidney lysosomal enzymes were examined. Among 34 pigmentation mutants which have been studied (Novak, E. K., Wieland, F., Jahreis, G. P., and Swank, R. T. (1980) Biochem. Genet. 18, 549-561) eight are known to have a 1.5- to 2.5-fold increase in kidney beta-glucuronidase in testosterone-treated females. These mutants appear to have defects in lysosomal processing, and because the mutations are at separate loci, each mutant probably affects different steps in assembly and/or exocytosis of lysosomes and related subcellular organelles. To test whether the neutral glycosphingolipids, galabiglycosylceramides, and globotriglycosylceramides thought to be associated with kidney lysosomes (McCluer, R. H., Williams, M. A., Gross, S. K., and Meisler, M. H. (1981) J. Biol. Chem. 256, 13112-13120) also exhibit abnormal secretion in the mutants with lysosomal enzyme abnormalities, the mutants beige-J, pale ear, light ear, pallid, and ruby eye-2-J were studied. The kidney and urine neutral glycosphingolipids from males of each mutant and C57BL/6J control mice were analyzed by high performance liquid chromatography. Beige-J, light ear, and pale ear showed marked increases in total kidney glycolipids; globotriglycosylceramides accounted for the bulk of the increase. Ruby eye-2-J showed less marked but significantly increased quantities of one galabiglycosylceramide and the globotriglycosylceramides in kidney. Pallid showed no significant increase in total kidney glycolipids but the globotriglycosylceramides appeared slightly elevated. In terms of the decrease in total urinary glycosphingolipids, the mutants fell into 2 categories. Beige-J, light ear, and pale ear were severely affected, whereas ruby eye-2-J and pallid were affected to a much lesser extent. Within the most severely affected group the excretion of the globotriglycosylceramides was more severely affected than that of the galabiglycosylceramides. The galabiglycosylceramides and globotriglycosylceramides appear to be specific markers of lysosomal membranes, but the independent behavior of these two classes of lipids during testosterone induction in normal mice and the differential effects on their secretion by different mutants indicate that they do not always exist in a characteristic ratio in a single type of subcellular organelle. All of the mutants accumulate organelles in their kidney proximal tubules which have distinct morphological characteristics as seen by electron microscopy.     

Distribution of murine stage-specific embryonic antigens in the kidneys of three rodent species

The distribution of two mouse stage-specific embryonic antigens (SSEA-1 and SSEA-3), recognized by monoclonal antibodies, was investigated in the kidneys of mice, rats and gerbils. Both antigens, with the exception of SSEA-3, which was not found in the kidney of the gerbil, were detectable in at least some portions of the kidney in all three species. These data suggest that while unique antigenic determinants may be evolutionarily conserved in homologous tissues of different species, their distribution may vary anatomically.     

Glucosylceramide in the androgen-responsive kidney of the lizard Anolis carolinensis

During the spring breeding season of the American chameleon, Anolis carolinensis, elevated levels of glucosylceramides which contain hydroxy fatty acids are produced in the kidneys of males but not females. Hyperproduction of this glycolipid is also induced by testosterone. The testosterone-induced hypertrophy of epithelial cells in the proximal tubules of the mouse kidney seems an analogous phenomenon and an elevated concentration of specific glycolipids in the male mouse kidney has been previously demonstrated. Thus the formation of renal glycolipids in response to testosterone may be a widespread feature in vertebrates.     

Biosynthesis of neutral glycosphingolipids in kidney slices from male and female mice

Previous reports from our laboratory (1981. J. Biol. Chem. 256: 13112-13120 and 1983. Endocrinology. 113: 251-258) showed the absence of Nfa-GalCer and Nfa-GaOse2Cer in kidneys of several strains of female mice. These lipids are always present in male kidneys and several other glycolipids are also elevated in males. To test whether this phenomenon is due to lowered biosynthesis in females, glycosphingolipid formation was assessed in kidney slices with [3H]galactose as precursor. The glycolipids were extracted after various incubation periods (from 30 min to 90 min) and individual glycolipids were separated and quantitated by high performance liquid chromatography and radioactivity was determined. The rate of formation of hydroxy fatty acid-containing galactosylceramide was the same in both sexes. The glycolipids which were low or not detectable in female kidney, Nfa-GalCer, Nfa-GaOse2Cer and Hfa-GaOse2Cer were rapidly labeled in the male kidney slices. These results suggest that nonhydroxy fatty acid-containing ceramide:UDP-Gal galactosyltransferase and hydroxy fatty acid-containing galactosylceramide:UDP-Gal galactosyltransferase have elevated activities in males. While the glucosylceramides are labeled at the same rates in both sexes, lactosylceramide appears to be labeled at higher rates in the male tissue. This suggests that glucosylceramide:UDP-Gal galactosyltransferase also has elevated activity in males. In addition, these data show that monohexosylceramides with different ceramide compositions are labeled at different rates.     

Age-related accumulation of autosomal mutations in solid tissues of the mouse is gender and cell type specific

Most cancers in solid tissues increase with age and invariably contain causal mutations eliminating expression of one or more autosomal tumor suppressor genes. However, very little is known about the effect of age on autosomal mutations, often large in size, in cells of solid tissues. In this study, the frequency and spectrum of autosomal mutations were examined as a function of age for kidney epithelial cells and ear mesenchymal cells in B6D2F1 mice heterozygous for the selectable Aprt locus. Aprt mutant frequencies were found to increase with age in the kidneys of both male and female mice, but at all ages the mutant frequencies were approximately twice as high in the females, which in this strain have shorter lifespans than the males. An age-related increase in Aprt mutant frequencies was also observed for ear cells from female mice, but no significant increases in mutant frequencies were observed for the ear cells of male mice. A molecular analysis showed that the kidney and ear mutational spectra were distinct and that the age-related increases in mutant frequencies did not involve significant shifts in the mutational spectra. In total, the results demonstrate both gender and cell-type-specific patterns of autosomal mutational accumulation as a function of age in two solid tissues of the mouse.     

Characterization of SSEA-1 glycolipids from the brain of a patient with fucosidosis

Neutral glycolipids from the brain of a patient with Fucosidosis were analyzed and two complex glycolipids containing five and eight sugars were isolated from the cortical grey matter. These two glycolipids reacted with antibodies recognizing the SSEA-1 [Le^x(X)] carbohydrate determinant. SSEA-1 glycolipids are normally expressed in human embryonic brain but are found in only small amounts in postnatal human brain. The accumulation of the two SSEA-1 glycolipids in Fucosidosis brain thus represents a defect which affects the normal developmentally regulated decrease in postnatal, expression of these glycolipids, and may be a contributing factor in the abnormal brain development associated with the disease. Chemical characterization of the two isolated glycolipids by gas chromatographic and mass spectrometric analyses has identified the two glycolipids as lacto-N-fucopentaosylceramide (III) and difucosyl-neolactonorhexaosylceramide.Abbreviations DCl direct chemical ionization - FAB tastatiom bombardment - GC gas chromatography - GSLs glycosphingolipids - MS mass spectrometry - SSEA-1 stage specific embryonic antigen-1 - TLC thin layer chromatographys     

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.     

Diabetic kidney disease in FVB/NJ Akita mice: temporal pattern of kidney injury and urinary nephrin excretion

Akita mice are a genetic model of type 1 diabetes. In the present studies, we investigated the phenotype of Akita mice on the FVB/NJ background and examined urinary nephrin excretion as a marker of kidney injury. Male Akita mice were compared with non-diabetic controls for functional and structural characteristics of renal and cardiac disease. Podocyte number and apoptosis as well as urinary nephrin excretion were determined in both groups. Male FVB/NJ Akita mice developed sustained hyperglycemia and albuminuria by 4 and 8 weeks of age, respectively. These abnormalities were accompanied by a significant increase in systolic blood pressure in 10-week old Akita mice, which was associated with functional, structural and molecular characteristics of cardiac hypertrophy. By 20 weeks of age, Akita mice developed a 10-fold increase in albuminuria, renal and glomerular hypertrophy and a decrease in the number of podocytes. Mild-to-moderate glomerular mesangial expansion was observed in Akita mice at 30 weeks of age. In 4-week old Akita mice, the onset of hyperglycemia was accompanied by increased podocyte apoptosis and enhanced excretion of nephrin in urine before the development of albuminuria. Urinary nephrin excretion was also significantly increased in albuminuric Akita mice at 16 and 20 weeks of age and correlated with the albumin excretion rate. These data suggest that: 1. FVB/NJ Akita mice have phenotypic characteristics that may be useful for studying the mechanisms of kidney and cardiac injury in diabetes, and 2. Enhanced urinary nephrin excretion is associated with kidney injury in FVB/NJ Akita mice and is detectable early in the disease process.     

Expression of glycosphingolipids in serum-free primary cultures of mouse kidney cells: male-female differences and androgen sensitivity

The expression of neutral glycosphingolipids was examined in primary kidney cell cultures derived from adult male and female beige mutant mice (C57BL/6J;bg ^j/bg ^j) with enrichment for proximal tubule cells during preparation of explants and using defined serum-free medium for the culture conditions. Cell proliferated for 7 daysin vitro to provide confluent or nearly confluent monolayers of epithelial-type growth indicative of proximal tubule cells. The malevs female differences in neutral glycosphingolipids seen in the kidneyin vivo were retained in these 7 day cultures. Cultures derived from males contained galacto- and digalactosylceramides whereas those from females did not express these types of glycolipids. Also, male cells had higher ratios of sphingosine: phytosphingosine containing species in Nfa (non-hydroxy fatty acid) globotriaosylceramide and in glucosylceramide than females. The shift in sphingosine: phytosphingosine to male ratios in Nfa globotriaosylceramide and in glucosylceramide could be stimulated in female kidney cells by treatment with 10^–5 M testosterone or 5-dihydrotestosterone. The male-specific expression of neutral glycosphingolipids, then, appears to be stable character of male-type differentiation in mouse kidney that is passed on during proliferation in culture. Female kidney cells retain an ability to respond to androgens with specific changes in neutral glycosphingolipid expression during 7 days of growthin vitro in serum-free conditions, but do not respond with the induction of the male-specific glycolipids galacto-and digalactosylceramides as seenin vivo.     

Dopamine production by the isolated perfused rat kidney

We used isolated perfused rat kidneys to examine dopamine (DA) production and its relation to renal function. Both innervated and chronically surgically denervated kidneys perfused with a solution containing neither albumin nor tyrosine, excreted 0.2 +/- 0.1 ng DA X min-1 X g wet weight-1 during the 10-min collection period between 30 and 40 min after starting perfusion. When perfused with 6.7% albumin, without tyrosine, innervated kidneys excreted 1.0 +/- 0.06 ng DA X min-1 X g-1 and denervated kidneys excreted 1.0 +/- 0.07 DA X min-1 X g-1. When 0.03 mM tyrosine was included in the albumin perfusate, innervated kidneys excreted 1.2 +/- 0.1 ng DA X min-1 X g-1 (p less than 0.1). Under these conditions DA excretion continued for at least 100 min at which time it was 0.6 ng X min-1 X g-1 and 86 ng/g kidney weight had been excreted. Denervated kidneys perfused with albumin + tyrosine excreted 0.9 +/- 0.13 ng DA X min-1 X g-1. Renal stores of free DA, conjugated DA, and dihydroxyphenylalanine (DOPA) could have provided at the most 30 ng/g of DA. Carbidopa inhibited DA excretion completely. DA excretion did not correlate with renal vascular resistance, inulin clearance, or fractional sodium excretion. In summary, nonneural tissue in isolated perfused kidneys produced DA at the same rate as denervated kidneys in vivo. Less than one-third of the DA produced by isolated kidneys could have come from intrarenal stores of DOPA, free DA, and conjugated DA; the rest was synthesized from unknown precursors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of immunoreactive napsin A in human urine

Human napsin A is an aspartic proteinase highly expressed in kidney and lung. To elucidate whether napsin A is excreted in the urine we have performed an immunochemical study using anti-napsin A polyclonal antibody. As a result an immunoreactive band at approx. 38 kDa was detected which corresponds to the molecular mass of recombinant active human napsin A. A deglycosylation study showed that excreted napsin A is N-glycosylated on apparently all of the three potential glycosylation sites. Immunoreactive napsin A was also observed in urine from patients with a transplanted kidney whose kidney function appeared half to fully normal. On the other hand, no or very low immunostaining was detected in samples from patients with diseased kidneys. The urinary excretion pattern correlates well with the enzymatic activity of napsin A. These data show that human napsin A is excreted as functional proteinase in the urine. Furthermore, immunochemical studies suggest a relation between urinary excretion of napsin A and renal function. More specifically, lack of urinary excretion of napsin A could potentially serve as a tool for the detection of kidney dysfunction.     

Excretion of water, sodium, and potassium during the compensatory renal hypertrophy in hypothyroid rats]

Renal compensatory hypertrophy is studied in age matched euthyroid and radiothyroidectomized female rats. 7 days after uninephrectomy, the hypertrophy of the remaining kidney is equally small in both groups. But 60 days after this operation, the hypothyroid animals show only a 12% increase in the wet weight of the remaining kidney whereas the euthyroid controls increase this weight by 21%. The excretion of water, Na and K are determined in the urine excreted in 5 h after a small water load. The results are related to 1 gram of kidney wet weight. These outputs increase in all animals after uninephrectomy. They are significantly higher in the hypothyroid rats than in the euthyroid controls as well before than 60 days after uninephrectomy. The reduction in tubular Na reabsorption found in the hypothyroid rat may account for the impairment of compensatory renal hypertrophy in hypothyroidism.     

Lysosomal mutations inhibit lipofuscinosis of the spleen in C57BL mice

Beige, bg, and reduced pigmentation, rp, are recessive mutations affecting lysosomal function. Homozygosity for beige prevented lipofuscinosis of the spleen in C57BL mice and its incidence was greatly reduced by homozygosity for rp. Dilute (d) homozygotes, with normal lysosomes, were susceptible to lipofuscinosis even though their melanosomes were more severely affected than those of beige mice.     

Genetic control of the expression of two extended globoglycolipids carrying either the stage specific embryonic antigen-1 or -3 determinant in mouse kidney

In the preceding paper (J. Biochem. 101, 553-562 (1987], we reported the structures of two neutral glycolipids (GL-X and GL-Y) purified from mouse kidney, and demonstrated the occurrence of polymorphic variation of these two glycolipids in inbred strains of mice. GL-X was characterized as galactosyl beta 1-3globotetraosylceramide, which was reported to be stage specific embryonic antigen-3 (SSEA-3) by Kannagi et al. (J. Biol. Chem. 258, 8934-8942 (1983], and GL-Y as 3-fucolactosaminyl beta 1-6(galactosyl beta 1-3)globotetraosylceramide, which carries the SSEA-1 determinant. In order to elucidate the mode of genetic control of GL-X and GL-Y expression, two variants, i.e., BALB/c mice expressing GL-Y and DBA/2 mice lacking GL-Y but expressing GL-X as the major neutral glycolipid, were subjected to mating experiments and glycolipid analysis. F1 hybrids expressed GL-Y, therefore GL-Y expression is dominant, and DBA/2 mice were determined to be recessive homozygotes. Through analysis of backcross and F2 mice, DBA/2 mice were demonstrated to carry a single defective autosomal gene, and it is concluded that because of this DBA/2 mice cannot express GL-Y and so accumulate GL-X. An attempt to map the gene controlling GL-Y expression on mouse chromosomes using coat colors and recombinant inbred strains was not successful.