THE JOURNAL OF B~LOGIC~L CAEMI~TRY Vol. 249, No. 10, Issue of May 25, PP. 3267-3272, 1974

Printed in U.S.A.

A Locus Determining P-Galactosidase Activity in the Mouse*

(Received for publication, November 2, 1973)

JAMES FELTON$, MIRIAM MEISLER,~ AND KENNETH PAIGEN

From the Department of Molecular Biology, Roswell Park Memorial Institute, Bufalo, New York l,JSOS

SUMMARY

A group of inbred mouse strains, typified by C3H/HeJ, have twice as much /3-galactosidase activity in their tissues as mice of a second group of strains represented by DBA/Z J. In all organs tested, the higher activity in C3H/HeJ is ex- pressed throughout development. The difference in activity is determined by a locus designated P-galactosidase (Bgs) on chromosome 9. The alleles present in C3H/HeJ (Bgsh) and in DBA/LiHa (Bgsd) show additive inheritance.

Studies of pH optima, heat lability, intracellular location, and molecular weight of the fl-galactosidase activity in crude homogenates suggest that only a single enzyme component is present. Partially purified enzyme preparations from C3H/HeJ and DBA/2J were indistinguishable with respect to heat lability, K, for fi-nitrophenylgalactoside, and molecu- lar weight. The presence of a single enzyme component, the absence of detectable structural differences, and the additive inheritance of Bgs alleles suggest that this locus may be either a structural gene duplication or a regulatory site con- trolling the transcription or translation of a closely linked fl-galactosidase structural gene.

The biochemical genetics of P-glucuronidase has been ex- tensively studied in the mouse, and genetic variants affecting the structure of the enzyme (l),’ its intracellular location (2), its control by steroid hormones (3), and its developmental pat- tern (4) have been described. However, we do not know to what estent the genetic controls affecting fi-glucuronidase are unique to this enzyme and to what extent they apply to other acid hydrolases. To approach this question, we have begun to define the genetic control of fl-galactosidasc activity. These studies have already shown that the activities of P-galactosidase and fl-glucuronidase are coordinately determined during develop- mcnt (5).

We have cl~osen P-galactosidase for these studies because abnormalitics in this enzyme have been implicated in a variety

* This research was supported by United States Public Health Service Grants GM 18484 and GM 19521.

1 Part of this work was included in the doctoral thesis of J. F. Present address, Room 5B-09, Building 10, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md. 20014.

0 Present address, Department of Biochemistry, State Uni- versity of New York, Buffalo, N. Y. 14214.

1 P. Lalley and T. Shows, personal communication.

of human congenital defects and we hope that information gained from animal studies of this enzyme will be of assistance in under- standing the human disorders.

In this report, we show that the common inbred strains of mice are readily divisible into two groups on the basis of their brain P-galactosidase levels. Mice of strain C3H, chosen to represent one of these groups, have twice the activity of C57BL/Ks and DBA/2 mice, chosen to represent the other group, in all organs tested at all stages of development. This difference is determining by a single locus on chromosome 9, with two alleles exhibiting additive inheritance. In a later report we shall describe a second variant in which only the liver activity is altered.

MATERIALS AND METHODS

Animals

Mice for the strain survey and for developmental studies were obtained from the Research Stocks and Production Department of the Jackson Laboratory, Bar Harbor, Maine. For genetic crosses, mice of strains DBk/LiHa (previbusly known as-DBA/ 2Ha (1)) and C3H/HeHa were kindly provided by Dr. T. S. Hauschka, Roswell Park Memorial Institute. Mice were main- tained on a 12.hour light, 12.hour dark schedule, and fed ad libitum on Rockland Mouse Breeder Diet (Teklab. Inc.. Mon- mouth, Ill.) containing 17% minimum crude protein, 10% mini- mum crude fat, and 2.5yo maximum crude fiber.

Tissue Homogenates

Freshly dissected tissues were weighed and homogenized in 0.25 M sucrose-O.02 M imidazole buffer. DH 7.0. (5 to 10%. w/v)

I _ I . 1-1 I

with a Polytron homogenizer (Brinkmann) for 1 min. Homog- enates stored at -20” retained their fl-galactosidase activity for 2 years.

Enzyme Assay

In our standard assay, reaction mixtures contained: 0.9 ml of 0.1 M citrate buffer, pH 3.5; 0.05 ml of 0.1 M p-nitrophenyl-fl-n- galactoside (Pierce); and 0.05 ml of tissue homogenates. Re- actions were started by addition of substrate, incubated for 30 to GO min at 37”, and stopped by cooling in an ice bath followed by rapid addition of 0.2 ml of 30% trichloroacetic acid. After centrifugation for 10 min to remove precipitated protein, the supernatant solutions were transferred to tubes containing 0.2 ml of 5.0 M 2-amino%methyl-1:3-propanediol (Aldrich). The absorbance of released p-nitrophenol was measured in a Zeiss spectrophotometer at 415 nm (E,, = 14,000).

The reaction rate was proportional to enzyme concentration and to time of incubation for more than 3 hours. Enzyme activity is expressed as micromoles of p-nitrophenol produced per hour.

Automated Assay of fl-Galactosidase

For analysis of the large numbers of animals generated in genetic crosses, an auto-analyzer was designed which measures hydrolysis of the fluorogenic substrate 4-methylumbelliferyl-&n-

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galactoside (Pierce) by small samples of tissue homogenates.2 The activity of murine p-galactosidase towards the 4-methyl- umbelliferyl derivative is approximately one-third of the ac- tivity toward the p-nitrophenyl compound. The 4-methylum- belliferylgalactoside assay was used exclusively to analyze the segregation of p-galactosidase activity in genetic crosses.

Presentation of Data

In graphing frequency distributions, we have indicated the number of animals whose activity falls into each class interval. The class intervals employed follow a logarithmic scale in which the upper limit of each interval is 1.06 times the value of the lower limit (0.025 log unit). We do so because the populations we have studied have relatively constant coefficients of variation (standard deviation/mean), but quite different means. The use of a logarithmic scale is advantageous in comparing such popula- tions since the coefficient of variation bears a constant relation- ship to the width of a class interval; as a result, the spread of each frequency distribution becomes independent of the absolute values of its mean. This is especially obvious in examining genetic crosses whose progeny segregate into distinct activity classes. The logarithmic scale avoids the artificial compression of the lower activity group, or the scatter of the higher group, _ - that attends the use of a l&ear scale.

_ - -.

The class interval emnloved here. 0.025 loe unit. is well suited for these purposes. In-addition to’ providilg an interval some- what smaller than the observed coefficients of variation, and hence useful in graphing moderate sized populations, it has the convenient property that 12 class intervals represent a doubling of activity and 40 class intervals a lo-fold increase.3

Protein

This was determined by the biuret method (6) with bovine serum albumin as standard. When necessary, turbid solutions were clarified after color development was complete by extraction with an equal volume of ether.

Enzyme Purification

Thirty-fold purification of mouse liver p-galactosidase was obtained by modification of a procedure for human liver p-galac- tosidase (7). All steps were carried out at 4”.

Step I-Livers from 12 to 18 mice were pooled and homogenized (2070, w/v) in 0.02 M phosphate buffer, pH 6.8, with a Polytron homogenizer for 2.5 min. Homogenates were frozen, thawed, and centrifuged for 30 min at 105,000 X 9.

Step d-The supernatant was diluted with an equal volume of buffer prior to the addition of ammonium sulfate (Mann ultra- pure) to a final concentration of 350 g per liter. Precipitated protein was collected by centrifugation at 105,000 X g for 60 min and redissolved in the original volume of buffer.

Step s--An equal volume of cold ethanol-acetone-ether, 15:4: 1, was added slowly with stirring. Precipitated protein was col- lected by centrifugation at 14,000 X g for 30 min and discarded. To the supernatant solution was added an additional 1.5 volumes of the organic solvent mixture. The precipitate was collected by centrifugation, resuspended in a small volume of 0.04 M sodium phosphate buffer (pH 6.8), frozen, thawed, and clarified by cen- trifugation at 105,000 X g for 30 min. The clear supernatant solution was enriched 20.fold for B-galactosidase activity with 90% recovery of the original activity.

Step &Additional purification was obtained by chromatography on Senhadex G-100. 120 mesh. in 0.04 M sodium ohosohate buffer. pH 6.k. The elutibn volume’ of P-galactosidase coriesponded td a protein with a molecular weight of approximately 80,000. Active fractions were pooled and concentrated by ultrafiltration in an Amicon cell, with UM 10 filter. The concentrated enzyme prep- aration was stable when frozen for 6 months.

Heat Inactivation

In order to study the temperature dependence of fl-galactosidase inactivation, a temperature gradient apparatus was constructed

2 K. Paigen and F. Pacholec, “Automated Assay of Glyco- sidases,” unpublished.

3 The same scale applied to acoustic frequencies is the basis for the modern la-note octave where it provides much the same utility.

from a solid aluminum block, 20 X 50 X 8 cm, which was milled at each end to provide a channel for circulation of water. The block was insulated on all sides with $5 inch of polystyrene. A temperature gradient was maintained along the length of the block by circulation of water through each end at two different temperatures. Eight rows of wells for test tubes, 15 mm in diameter X 60 mm in depth. were drilled alone: the lenzth of the block. Tubes containing enzyme were incubated in these wells for 10 min. The temperature of each tube was measured with a Digitec digital thermometer.

Electrophoresis

Electrophoresis was performed in Tris-glvcine buffer at DH 8.1 as previo&ly described (5). Seven per cent polyacrylamihe gels (5 X 75 mm) were nrerun for 15 min at 0” and 200 volts. Samnles I acre subjected to electrophoresis for 15 min at 200 volts and then 60 min at 300 volts (about 2 ma per tube). After removal from running tubes, the pH of the gels was adjusted by incubation for 30 min in 0.1 M citrate, pH 3.5. o-Galactosidase activity was visualized with a staining solution (8) prepared as follows: 15 mg of 5.bromo-4.chloro-3-indoyl-P-n-galactoside (Sigma) was dissolved in 0.6 ml of dimethylformamide, followed by the se- quential addition of 24 ml of 0.1 M citrate buffer (pH 3.5), 0.3 ml of 0.85% NaCl, 6 mg of spermidine HCl, and 2.25 ml of a mix- ture containing equal volumes of 0.05 M potassium ferricyanide and 0.05 M potassium ferrocyanide. The staining solution was stable for 2 days. After gels were stained at 37” for 30 to 60 min, fl-galactosidase appeared as a blue-green band.

RESULTS

Strain Variation-A set of 45 inbred mouse strains, chosen to provide as diverse a genetic pool as practicable, was surveyed for genetic variants of @-galactosidase. In order to detect struc-

tural variants, the electrophoretic mobility and thermal sen-

sitivity of the enzyme were tested. To detect variants with altered intracellular location of enzyme, the relative amounts of

soluble and particulate activity were assayed in liver. To detect regulatory and developmental variants, the quantitative levels of enzyme activity in a set of adult tissues were compared. No mutants in structure or subcellular location were detected among the set of 45 strains. However, we did find considerable varia- tion in total enzyme activity among adult organs.

The strains could readily be divided into two groups on the basis of their brain enzyme activity. The members of one group show brain enzyme activities of 6 to 9 pmoles per hour per g and those of the other group 13 to 16 pmolcs per hour per g. Brain activity was chosen as the distinguishing characteristic because there is very little individuai variation in this activity, and because the distinction between the two groups is unam- biguous (Fig. 1). The values of brain fl-galactosidase for these inbred strains is presented in Table I.

We have chosen one strain from the high activity group, C3II/HeJ, and two strains from the low activity group, DISA/2J and C57BL/Ks, for further comparison. The value of P-ga- lactosidase specific activity in adult tissues of C3H/HeJ mice is twice as high as the corresponding DEA/2J tissues (Table II).

6 8 IO 12 14 16

/3-GALACTOSIDASE SPECIFIC ACTIVITY

FIG. 1. Distribution of brain P-galactosidase among inbred strains of mice. Each point represents one strain; the strains are identified in Table I.

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TABLE I TABLE II

Variation in specific activity of brain p-galactosidase among inbred strains of mice

Enzyme activity and protein concentration were determined on homogenates prepared from pooled tissues of three adult males from each strain.-

@-Galactosidase activity in CSHjHeJ and DBA/$J mice -Organs from 12 mice of each strain were individually homoge-

nized and assayed. Activity is expressed as the mean micromoles per hour per g f 1 S.E.

Tissue DBA/2J C3H/HeJ C3H:DBA

Low Class

Strain

-

1

-

tiicrometers per hour

Per g Strain

b

.-

AKR/J 6.6 A/HeJ 14.4 AU/SsJ 7.0 A/J 13.9 BAN/Re 6.8 BALB/c 13.3 BDP/J 7.0 CE/J 15.1 BUB/BnJ 7.0 C3H/HeJ 15.1

CBA/J 7.7 C57BL/6J 13.5 CBA/&J 7.1 C57BL/lOJ 14.8 C57BL/KsJ 6.4 C57Br/cdJ 15.1 DBA/lJ 5.7 C57e/Ha 13.4 DBA@J 6.5 C57L/J 14.4

I/FnLn 8.9 C58/J 14.9 LG/J 8.8 DE/J 16.0 LP/J 8.8 DW/J 16.3 NZB/BlN 7.8 FL/lRe 15.1

P/J 7.4 HRS/J 12.7

PL/J 6.8 MA/J 15.8 RF/J 6.6 MK/Re 14.9 RIII/BJ 6.8 PH/Re 14.4 SEC/lReJ 7.7 WB/Re 15.1 SJL/J 5.6 YBR/WiHa 15.7

SM/J 7.7 ST/bJ 8.3 SWR/J 9.3 WC/Re 7.5 WH/Re 7.5

WK/Re 8.0 129/J 8.1

-

-

- High Class

a The data have been corrected for a 50/O interstrain variation in protein concentration: the values are micromoles per hour per g wet weight divided by the factor:

protein concentration of specific strain average protein concentration of all strains

Mixtures of samples from the 2 strains have the predicted addi- tive activity, indicating that low molecular weight effecters are not responsible for the activity difference.

DeveZopment-The higher activity in C3H/HeJ relative to that in DBA/2J and C57BL/Ks does not appear to arise from a difference in the developmental pattern of the enzyme in these strains. Enzyme activities were measured in brain, liver, and heart of mice of each strain between gestational Day 14 and 60 days of age (Fig. 2A). The higher activity of C3H was main- tained throughout this period, the activity in C3H being almost exactly twice that of the other strains in each tissue at all stages of development (Fig. 2B).

Enzyme Properties-The characteristics of the fi-galactosidase activity present both in crude homogenates and in partially purified preparations from C3H/HeJ and DBA/SJ livers were compared to determine whether the difference in activity re-

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Brain. . . . . 7.6 f 0.1 16.4 f 0.1 2.1 Liver . . . . . . . . . . . 14.9 f 0.5 29.8 f 0.9 2.0

Heart.. . . . . 4.2 f 0.2 7.2 f 0.1 1.7 Kidney. . . . 71.0 f 1.4 148.0 f 2.4 2.1 Spleen. . . 49.0 & 1.6 120.0 f 3.5 2.4

3J- 20 I

A B

2- -10 0 IO 20 30 40 50 -10 0 1020304050

AGE IN DAYS

FIG. 2. &Galactosidase activity in developing mouse organs Mice were obtained from the Production Deoartment of the Jackson Laboratory and tissues were prepared~as described (5) Birth was on Day 0. Liver, brain, and heart were studied in on high activity strain (C3H/HeJ (O-0)) and in two low activit: strains (DBA/BJ (O-O) and C57BL/Ks (O---O)). A activities per g of tissue. B, relative activities. The data arl expressed relative to the average value at each time point calcu lated from the DBA/B, C57BL/Ks, and one-half of the C3I activities.

fleeted any difference in the properties of the enzyme. Severa lines of evidence suggest that a single enzyme component i responsible for the P-galactosidase activity present in the tissue we have studied.

About 85% of the total &galactosidase activity of liver wa estimated to be in lysosomes from measurement of the relativ amounts of soluble and particulate activity and the observation

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I /322 l/318 l/314

I/K0

FIG. 3. Thermal inactivation of mouse &galactosidase. Puri- fied enzyme preparations from C3H/HeJ (O-O) and DBA/BJ (0-O) livers were incubated in 0.1 M citrate buffer, pH 3.5, for 10 min at the indicated temperatures. Residual enzyme activity was then determined under standard assay conditions. Rates of inactivation were calculated with the assumption that denaturation followed first order kinetics; this was confirmed in control experiments.

that nearly all of the particulate activity was released when the particulate fraction was exposed to hypotonic buffer (9). There was no difference between strains in this regard. The enzyme from the two strains had the same elution volume after gel filtration (see “Materials and Methods”).

In every case the kinetics of heat denaturation of the activity present in crude homogenates was monophasic. There was no indication of more than one enzyme component. The sensitivity to thermal denaturation was measured over the range 37-49” for partially purified enzyme (Fig. 3). From these data, a value of 39,000 cal per mole for the energy of activation of the denatura- tion reaction was calculated for both strains. The kinetics of denaturation was tested at 43” and was first order with a rate constant of -0.07 min+ for both strains.

We have had considerable difficulty in carrying out acrylamide gel electrophoresis. Single bands with similar mobilities in all strains were observed at pH 8.1; however, the staining reaction requires high protein concentrations and the resolution was poor.

The enzyme present in both C3H/HeJ and DBA/SJ liver has optimal activity at pH 3.5, and there was no difference in the pH activity curves of the two strains. Enzyme from both strains is completely inhibited by lop5 M p-chloromercuriben- zoate. The affinity of the enzyme for the substrate p-nitro- phenyl-fl-n-galactoside was determined (Fig. 4). A value of 5.2 x 10m4 M was obtained for the K, of enzyme preparation from both strains.

Taken collectively, these results suggest that a single enzyme component is present in both strains, and that the difference in &galactosidase activity between C3H and DBA/S does not result from a change in either the intracellular location or struc- ture of this enzyme.

Genetics-The difference in activity between C3H and DBA mice appears to be controlled by a single locus with two alleles showing additive inheritance. The brain P-galactosidase ac-

OL----- 2 4

G16

0 IO

x 10-4

FIG. 4. Affinity of @-galactosidase from C3H/HeJ (O-O) and DBA/SJ (O---O) livers for the substrate p-nitrophenyl-&n- galactosidase. The apparent K, for enzyme from both strains is 5.2 X 10-* M. The data are plotted using the Eadie-Hofstee (10) linear transform of the Michaelis-Menten equation.

TABLE III Linkage of Bgs locus with dilute locus

Eighty offspring of the reciprocal backcrosses between (C3H/ HeHa X DBA/LiHa)FI mice and the DBA parent were scored for coat color and for brain fi-galactosidase. The segregation of enzyme levels into low and intermediate classes is evident in Fig. 5. The substrate used was 4-methylumbelliferyl-P-n-galactoside. The activity against this substrate is lower than with p-nitro- phenyl-fl-n-galactoside.

Brain p-galactosidase

Coat color Low (1.8-2.8

pmoles/hr/g)

Intermediate (3.6-5.2

pmoles/hr/g) Total

I I I

Dilute. . . . Nondilute Total.. . . .

tivity of F1 progeny of a cross between C3H and DBA showed intermediate levels of activity. Progeny of the backcross of the F1 to the C3H parent segregated into the intermediate and high classes in approximately equal numbers (Fig. 5). Similarly, progeny of the backcross to the DBA parent segregated into the intermediate and low classes in approximately equal numbers (Fig. 5). The Fz generation segregated into the expected three classes. The locus has been designated Bgs (fl-galactosidase) ; the allele present in C3H/HeJ is Bgsh and that present in DBA/ 25 is Bgsd.

Linkage tests to known loci (Table III), showed Bgs to be approximately 21.2 f 4.6 centimorgans from the chromosome

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3271

C3t-i /HeHa

ii

DBA/Li Ha

Iii .

F, i

. . II ii . F2 , A .llll~l*r.,i,* . ..I l *e

Backcross : . to C3H

Backcross to DBA

.

,I, ,,,,,I * ,,,,,,,,,,,,,.,,,,, ,,,,,,,,,

I.20 I.50 2.00 2.5D 3.16 4.00 SIX) 6.30 6.00 IO.00

Brain B- Galactosidose Activity

FIG. 5. Inheritance of p-galactosidase activity. Brain enzyme activity was determined for the parental strains C3H/HeHa and DBA/LiHa, and for the I?,, Fz, and both backcross generations. In all cases, reciprocal crosses were made, with no significant difference in the results. p-Galactosidase was determined by automated assay with the substrate 4.methyluInbelliferyl-/3-o- galactoside. All animals were between 9 and 12 weeks of age. Each filled circle represents one animal

9 marker dilute. This agrees with the observations of Hakansson and Lundin.

Levels of P-galactosidase in liver and brain appear to be deter- mined by the same locus. Animals segregating into the three brain activity classes, low, intermediate, and high, simultane- ously segregate into the corresponding classes of liver activity (Fig. 6).

DISCUSSION

Inbred mouse strains show significant differences in levels of fi-galactosidasc; all of the strains examined could be classified as high fl-galactosidase (specific activities between 13 and 16 ~rnoles per hour per g wet. weight of brain tissue) or low P-ga- lactosidasc strains (6 to 9 pmoles per hour per g). In a represent- ative high strain (CSH/HeJ), the P-galactosidase activity in all tissues at all stages of development is twice that of the low strains DlU/BJ and C57BL/Ils. In genetic crosses between these strains, this quantitative difference segregated as a single Mendelian factor. Linkage tests demonstrated that the re- sponsible locus, designated Bgs, is located on chromosome 9 in the mouse, approximately 21 ccntimorgans from the dilute locus. The enzyme level in heterozygotes was equal to the mean of the parental activities, i.e. the locus exhibits additive inheritance.

. .

.

.

2

t I I I I I I J

0 I 2 3 4 5 6 7

BRAIN P- GALACTOSIDASE ACTIVITY

FIG. 6. Coordinated segregation of p-galactosidase activity in brain and liver. Activity was determined in both tissues of each animal in the backcross generations described in Fig. 5. Units are micromoles of 4-methylumbelliferyl-P-n-galactoside hy- drolyzed per hour per g of tissue. Backcross to DBA (0-O); backcross to C3H (O---O).

Two alternative models for the effect of the Bgs locus may be considered. First, the Bgsh allele (in C3H mice) may encode a structurally altered enzyme whose catalytic activity per mole- cule is twice as great as the enzyme encoded by the Bgs” allele (in DBA/2 mice). Alternatively, the effect of the Bgsh allele may be to increase the concentration of enzyme molecules in the tissues of C3H mice. The first model depends upon a dif- ference in primary structure between the fl-galactosidases present in C3H and DBA/2 mice, while the second model predicts no difference in structure. To test these predictions we compared the heat lability, electrophoretic mobility, and kinetic properties of fl-galactosidase from the two strains; no differences in these parameters were observed. The probability that a structural change would be reflected in one of these parameters can be estimated. Empirically, 70% (39/55) of amino acid sub- stitutions were found to alter the heat lability of bacterial /% galactosidase (II), and 67 y0 (14/21) of variants in activity of glucose g-phosphate dehydrogenase exhibited altered K, for substrate (12). Approximately 30 y0 of base substitutions the- oretically should produce a protein with altered net charge; however, we are not certain that a single charge difference would have been detected in our electrophoretic system. We feel that the absence of detectable differences in these three properties make it unlikely that the @-galactosidases of C3H/HeJ and DBA/2J mice differ in their primary structures and hence in their catalytic activity per molecule. However, definitive evi- dence on this point will require the purification of the enzyme and more detailed structural comparisons.

It appears more likely that the Bgs locus controls the number of enzyme molecules present in mouse tissues. Two models which account for the failure to detect any structural change and for the additive inheritance of the Bgs alleles are that Bgsh represents a duplication of the P-galactosidase structural gene, or that it represents an alternate form of a regulatory site con- trolling the efficiency of transcription or translation of a closely linked fi-galactosidase structural gene.

In anticipation of further studies of the Bgs locus we wish to 4 E. Hakansson and L. Lundin, personal communication, 1973. point out that all presently reasonable models for its action in-

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3272

dicate that Bgs is either identical with or closely linked to the structural gene for @-galactosidase.

In considering possible models of genetic regulation in mam- mals, it is of considerable interest that while fl-galactosidase and fl-glucuronidase are both present in lysosomes, and share a coordinated pattern of development (5), the genetic factors con- trolling their expression are not associated physically on the same chromosome; the Bgs locus is 011 chromosome 9, and the P-glucuronidase structural gene with its attendent regulatory sites is 011 chromosome 5 (l-4, 13).’

Acknow2edgments-We are grateful to Dr. Douglas Coleman of the Jackson Laboratory, Bar Harbor, Maine, for sponsoring our studies at the Jackson Laboratory during the summer months of 1970 to 1972. The st,rain survey and developmental studies

were great,ly facilitated by the cooperation of the Laboratory staff.

REFERENCES

1. PAIGEN, K. (1961) Exp. Cell Res. 26, 286 2. GANSCMOW, It., AND PAIGEN, K. (1968) Genetics 69, 335 3. SW.~NK, R..T.,.AND PAIGEN,.K. (1973) J. Mol. Biol. ‘77, 371 4. PAIGEN. K. (1961) Proc. Nat. Acad. Sci. U. S. A. 47. 1641 5. Mm& M:, AND PAIGEN, K. (1972) Science

LAYNE, 6. (1957) Methods Enzymol. 3, 447 177, 894

6. 7. MEISLER, M. (1972) Methods Enzymol. 28, 820 8. YARBOROUGH, D., MEYER, O., DANNENBERG, A., AND PEAR-

SON, B. (1967) J. Reticuloendothelial Sot. 4, 390 9. GANSCHOW, It., AND PAIGEN, K. (1967) Proc. Nat. Acad. Sci.

U. S. A. 68, 938 10. HOFSTEE, B. H. J. (1952) Science 116, 329 11. LANGRIDGE, J. (1968) J. Bacterial. 96, 1711 12. YOSHIDA. A. (1973) Science 179. 532 13. SIDMAN, k. L.; AND GREEN, M. C. (1965) J. Hered. 66, 23

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James Felton, Miriam Meisler and Kenneth Paigen-Galactosidase Activity in the MouseβA Locus Determining

1974, 249:3267-3272.J. Biol. Chem. 

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