GENOMICS

11,1054-1062

(1991)

Molecular J. M. *Howard

FRIEDMAN,*‘t

Hughes Medical

Institute,

Mapping R. L.

LEIBEL,$

of the Mouse ob Mutation D.

s. SIEGEL,*‘t

AND

N.

BAtiARYt

tlaboratory of Molecular Cell Biology, and *Laboratory of Human Behavior and Metabolism, The Rockefeller University, New York, New York 1002 1 Received

April 4, 1991;

revised

July 15, 1991

mentation of this approach will provide insight into the molecular mechanism(s) by which a single gene product can act to regulate (suppress) body weight. Our initial objective has been to segregate the ob mutation among -750 informative meioses. Toward that end, separate intraspecific and interspecific crosses segregating ob have been established. The progeny from these crosseshave been typed for a series of RFLPs from proximal mouse chromosome 6 to generate a molecular genetic map of the region around the ob locus. In addition, detailed phenotypic analyses of the obese (ob/ob) progeny reveal subtle but important phenotypic differences in body weight, fatness, and the development of type II diabetes among the progeny of each cross. Finally, characterization of these animals has also allowed us to compare the genetic maps in this region derived from interspecific and intraspecific crosses.

The mouse ob mutation has been mapped relative to a series of RFLPs among the progeny of three separate mouse crosses: an intraspecific backcross, an intraspecific intercross, and an interspecific intercross. Genotypic assignment at the ob locus was made by making use of measurements of body mass index and the plasma concentrations of glucose and insulin. These data have suggested that the development of diabetes in these animals is a consequence of unlinked polygenes. There was also evidence that unlinked i%fus spretu alleles can diminish the obesity of ob/ob mice. From these data we have mapped several markers on chromosome 6 with the following order: cenCola-2-Met-ob-Cpa-Tcrb. The homologs of markers that flank ob map to human chromosome 7q, suggesting that if o 1991 there is a human homologue of ob, it maps to 7q31. Academic

J. WALSH,*rt

Press, Inc.

INTRODUCTION MATERIALS

Obese (ob) is an autosomal recessive mutation on mouse chromosome 6 that occurred spontaneously in the mouse colony at the Jackson Laboratory (Ingalls et al., 1950). Homozygous animals develop profound and progressive obesity that is first detectable at 3 weeks of age. By age 6 months, mutant animals often weigh 80-90 g (80% fat) compared to 30 g (10% fat) for wild type littermates (Johnson and Hirsch, 1972). In addition, ob/ob animals manifest hyperphagia, hyperinsulinemia, and increased metabolic efficiency (weight or fat gain per calorie ingested) (Johnson and Hirsch, 1972; Coleman, 1978). The similarity of this metabolic phenotype to that in some obese humans has made these mice the subject of intense investigation (Leibel et al., 1990). Attempts to identify the ob gene product have been confounded by the complex secondary metabolic changes that occur in mutant animals and by an inability to determine the anatomic site(s) of expression of this gene (Coleman, 1982). We are endeavoring to clone the mouse ob gene using the techniques of positional cloning (Riordan et al., 1989). The successful impleoSSa-7543/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form

METHODS

1. Genetic Crosses The schemata for three genetic crosses segregating the ob mutation are shown in Fig. 1. Initially, we segregated ob among the progeny of an interspecific cross using C57BL/6J and Mus spretus. In this cross, B6 spretus ob/+ Fl females and B6D2 ob/+ Fl males were obtained by mating C57BL/6J ob/ob ovarian transplants to either lks spretus or DBA/BJ male mice (Fig; 1C). The ovarian transplants were kindly provided by Dorcas Corow. These Fl progeny were intercrossed, and the distance between a given RFLP and ob was determined by scoring the number of obese (ob/ob) F2 animals carrying either the spretus and/or DBA allele. Only obese F2 animals could be used for mapping in this cross because ob/+ and +/+ lean animals cannot be distinguished by phenotype. It should be noted that each obese F2 animal has two storable meioses;the male meiotic event scores recombination between C57BL/6J and DBA/BJ and the female meioses score recombinations between C57BL/6J 1054

Inc. resewed.

AND

MULEXULAK

MAl’Jm’1Nt.i

Ul’

and M. spretus. In three allele systems in which the C57BL/6J, DBA/PJ, and M. spretus alleles differ, male and female meiotic events can be scored independently. The met oncogene defines a three-allele system when the DNAs are digested with BgZII and in this case male and female meioses were scored independently. A total of 130 informative meioses with Mus spretus were scored using this cross. An equal number of informative meioses can also be scored for inheritance of the DBA allele although only 124 of the animals were scored for Met. Two additional crosses were also established: an intraspecific backcross and an interspecific intercross. In the former, C57BL/6J ob/ob X DBA/BJ Fl males were backcrossed to C57BL/6J ob/ob ovarian transplants (Fig. 1A). A total of 96 obeseand 111 lean progeny of this cross totaling 207 meioses were characterized. In the intraspecific intercross C57BL/6J ob/ob X DBA/W Fl males and females were mated (Fig. 1B). As was the case for the interspecific cross, only ob/ob progeny were used for genetic mapping. One hundred forty-seven obese intercross progeny totalling 294 meioses were generated from this cross and included in the mapping panel.

2. Animals All animals were weaned and separated by sex at about 21 days of age. Diet consisted of ad libitum Pica laboratory mouse breeding chow No. 5058 (Purina), containing 9% fat, and water. Animals were fasted for 15 h prior to sacrifice by CO, asphyxiation. Approximately 1.0 ml of whole blood was withdrawn by cardiac puncture from each mouse and added to 50 ~1 of 82 n&f EDTA as anticoagulant. Plasma was decanted after centrifugation and frozen at -80°C for subsequent assay of insulin (Herbert et aZ., 1965) and glucose (Kadish et al., 1968) concentrations. Kidney, liver, spleen, tail, and thoracic contents were immediately frozen at -80°C for subsequent isolation of DNA.

3. DNA Extraction

and Southern

Blotting

High-molecular-weight DNA was prepared from these organs by the method of Amar et al. (1985). Approximately 10 pg of DNA from each animal was digested with restriction endonuclease (Bethesda Research Laboratories Life Technologies, Inc., Gaithersburg, MD), electrophoresed in 1% agarose, and transferred to GeneScreen Plus nylon membranes (NEN Research Products, Boston, MA) by Southern blotting using 0.4 N NaOH/O.G N NaCl for transfer (Bahary et al., 1990).

L’HE

MUUSir;

00

1055

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4. Probes The probes used included those for T cell receptor /3

(Tcrb) (Snodgrass et al., 1985), carboxypeptidase A (Cpa) (Quint0 et aZ., 1982), met oncogene (Met) (Dean et al., 1987; gift of mouse met cDNA from George van de Woude), and a2 procollagen (Cola-2) (Liau et aZ., 1985). Inserts were purified from a low-melting-point agarose gel and labeled using an oligo priming reaction (Feinberg and Vogelstein, 1983). RESULTS

1. Genetic Crosses We have made use of C57BL/6J ob/ob ovarian transplants to establish an interspecific intercross, an intraspecific backcross and an intraspecific intercross. Lean and obese progeny of the backcross and obese progeny of the two intercrosses have been used to build a genetic map of proximal chromosome 6. The method by which we assigned genotype at the ob locus is discussed below. The use of an interspecific cross facilitates genetic mapping, i.e., the extensive polymorphism of M. spretus has made these animals useful for screening new markers (Avner et al. 1982). The inefficiency in breeding M. spretus has precluded the use of these mice to generate large numbers of obese animals. Thus, the progeny of two intraspecific crosses will be used for the detailed genetic mapping that will be necessary for the cloning of the gene. The three crosses are shown in Fig. 1.

2. Assignment

of Genotype at the ob Locus

A critical issue in all of these crosses is ensuring that the correct genotypic assignment at the ob locus is made in F2 or N2 animals. Generally, phenotypic differentiation between ob/ob and lean (ob/+ and +/+) animals is obvious on physical inspection (Fig. 2). However, to ensure unambiguous assignment of genotype at the ob locus, measures of body weight, body length, and plasma concentrations of glucose and insulin were made in each animal. Body weight alone cannot be used for distinguishing lean from obese animals since there is often overlap in the distributions of this parameter for ob/ob and wild type animals (Bahary et al., 1990). Such overlap reflects the effects of an unspecified number of unlinked alleles from the parental strains on body size and composition as well as on the phenotypic penetrance of the ob/ob genotype. In order to ensure that genotype was correctly assigned in all animals used in the preparation of these maps and to maximize the number of animals that could be scored for this purpose, we employed a protocol similar to that recently used in the molecular mapping of the mouse

1056

FRIEDMAN

B 04

C57BLEJ oblob transplant

AL.

::

6

A

ET

B

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C57BL/6J oblob

DBA/PJ +I+ males

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C57ELi6J

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w

ob C57BLXJ oblab tEXlE.pl~~t

I-

lean obese Parenta&

C57ELkJ x DBAiSJ ob/+ males

obese kIecombina&

lean

RFLP haplotypes, and phenotypes, of N2 animals resulting from: C57BU6J ob/ob X B6D2 Fl ob/+

C57BhJ DBA/PJ ob/+

x

c&se LParentad

C57BLk-J DBA/PJ ob/+

&as6

x C57Bb6; x M. saretus ob/+ females

t

C57BU6J x DBAIPJ obl+ males

-.

LRecombinants

““_I

RFLP haplotypes. and phenotypes. of F2 animals resulting from: B6D2 Fl oW+ x B6D2 Fl ob/+

Parental RFLP haplotypes from: B6D2

’

I Recombinants for obese progeny Fl ob/+ X B6spretus

(F2) resulting Fl ob/+

FIG. 1. Genetic crosses. The mouse ob gene was segregated in three different genetic crosses. Ovarian transplants make use of donor C57BL/6J ob/ob ovaries into recipient agouti (A”) females. This allows the use of coat color in Fl animals (except B6 spretus) to determine whether the Fl is derived from the donor ovary. (A) Intraspecific backcross. Wild type DBA male mice were mated to C57BL/6J ob/ob ovarian transplants. Fl males (B6D2 ob/+) were backcrossed to the ovarian transplanted mice to generate N2 animals. Both obese (ob/ob) and lean (ob/+) animals were used for genetic mapping. (B) Intraspecific intercross. Fl (B6D2 ob/+) female and male progeny of a DBA x C57BL/6J ob/ob cross were mated. Obese animals were collected and used for genetic studies. (C) Interspecific intercross. Mus spretus males were mated to C57BL/6J ob/ob ovarian transplants. Fl females (B6 spretus ob/+) from this cross were bred to Fl males (B6D2 ob/+) generated from a C57BL/6J ob/ob x DBA 25 +/+ mating. Only obese animals were used for genetic mapping.

diabetes (db) mutation (Bahary et al., 1990). db is an autosomal recessive mutation on chromosome 4 that produces a phenotype indistinguishable from ob/ob if bred on the same strain background (Coleman, 1978). This protocol for assignment of genotype at the ob locus makes use of body mass index (BMI = weight in grams/(nose-to-anus length in centimeters2) as the primary indicator of the ob genotype with secondary use of plasma concentrations of glucose and insulin to confirm the initial genotype assignment. BMI is often

FIG. 2. ob/ob and lean animals. Male sibling N2 animals from the intraspecific backcross are shown. The ob/ob animal weighed 79 g and his lean brother weighed 27 g. Reprinted courtesy of NEWSDAY/Ozier Muhammad.

used as an indicator of adiposity in humans (Keys et al., 1972), and this proportion also works well in distinguishing obese from lean mice (Bahary et al., 1990). As a preliminary indication of phenotype, a stem-leaf plot of BMI was prepared for each of the three crosses (Fig. 3, top panel). Each plot was visually inspected for a region of separation between the distributions for obese and nonobese animals. On this basis, the animals were separated into high-BMI (tentative ob/ob) and low-BMI groups (tentative wild type). The respective BMI cut points for lean and obese in the three crosses were interspecific intercross, to.407 = wild type, >0.417 = ob/ob; intraspecific backcross, co.387 = ob/+, ~0.462 = ob/ob; and intraspecific intercross, CO.397 = wild type, >0.400 = ob/ob (Table 1 and Fig. 3). The plasma concentrations of glucose and insulin were then used to confirm the genotypic assignments of high-BMI animals as ob/ob and low-BMI animals as wild type. These parameters are useful because ob/ ob animals are highly insulin resistant (but normoglycemic) during the first 4-6 months of life (Coleman, 1978). Thereafter, depending upon the genetic background on which these genes are carried, varying degrees of pancreatic /3 cell necrosis develop (Hummel et al., 1972). This eventuality is accompanied by declining plasma concentrations of insulin and the development of overt diabetes mellitus with elevated levels of plasma glucose. Thus, all ob/ob animals should have

MOLECULAR

MAPPING

OF

THE

MOUSE

ob

1057

MUTATION

N2

F2 SPRETUS

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BMI FIG. 3. Phenotypic characterization of obese and lean progeny. Top: histograms showing the number of animals with a particular body mass index (BMI) = wt/liter’ are presented for each of the three crosses. N2 represents the backcross, F2 DBA represents the intraspecific intercross and F2 spretus represents the interspecific intercross. A, female; B, male. Middle: Plasma insulin concentration was measured for each animal in each of the crosses and plotted vs BMI for that animal. Bottom: Measurements of plasma glucose concentration were also plotted relative to BMI for each animal. m, male; f, female.

1058

FRIEDMAN

either an elevated plasma concentration of glucose or insulin (or both). Measurements of these parameters were made on all F2 and N2 animals. To assign genotype at the ob locus the mean values and standard deviation of plasma glucose and insulin were calculated for the high- and low-BMI progeny of each of the three crosses. These data are shown in Table 1. Comparisons of plasma glucose and insulin levels relative to BMI for each animal are also shown in Fig. 3. The variance of plasma glucose and insulin concentrations notable in this figure also has implications for the inheritance of Type II diabetes in these animals and is discussed below. High-BMI animals that did not have either a plasma glucose or an insulin concentration three standard deviations higher than the mean value of the low-BMI group were considered to be nonobese and were not included in the mapping panel. Similarly low-BMI animals whose plasma glucose or insulin were three standard deviations higher than that of the low-BMI group were considered to be possible ob/ob animals and were also excluded from the mapping panel. The characteristics of six excluded backcross animals with low BMI but with high glucose and/or insulin levels are shown in Table 2. In summary, high-BMI animals with elevated concentrations of plasma glucose and/or insulin were considered to be ob/ob and low-BMI animals with low values for these parameters were considered to be nonobese (+/+ or ob/+). The cutoff points for glucose and insulin that were used to confirm these genotype assignments are also shown in Table 1. Comparison of the ob/ob and control (ob/+ or -t/+) progeny of each of the three crosses demonstrated no overlap in any of the parameters discussed above (data not shown). It appeared from this analysis of the backcross that ob is fully penetrant when bred to DBA (105 ob/+ vs 95 ob/ob). Similar calculations were not made for the intercrosses since we did not phenotypically characterize all of the low-BMI animals and are therefore uncertain as to the number of lean progeny from these crosses. We also compared the BMI, plasma glucose, and plasma insulin concentrations among the progeny of the three crosses. Of interest to us was the fact that differences in these parameters between the progeny of the interspecific and intraspecific intercrosses are solely a consequence of substituting hfus spretw alleles for DBA in the female parent (see Fig. 1). Comparisons between the progeny of the two intercrosses revealed a lower BMI among the ob/ob progeny of the interspecific cross. Progeny of the intraspecific intercross had an average BMI of 0.598 vs an average BMI of 0.551 for the ob/ob progeny of the interspecific cross (P < 0.0001). Thus, inheritance of Mus spretus alleles tended to diminish adiposity among the ob/ob F2 animals. Lean animals from the three crosses did

ET

AL.

TABLE Phenotypic Characteristics Animals (F2 and N2) in (Grouping Based on BMI)

1 of All Obese and Lean Each of Three Crosses

Cross A. Intraspecific

backcross

Probable genotype BMI category [Insulin] (SEM) Range Cut point [Glucose] @EM) Range Cut point BMI (SEM) Range B.

Intraspecific

intercross

Interspecific

Probable genotype BMI category [Insulin] (SEM) Range Cut point [Glucose] (SEM) Range Cut point BMI @EM) Range

(B6D2

ob/+

F16 X B6 ob/ob

lob/+) to.387 (n = 111) 46.8 (4.3) 1.2-277.6 65.1 234 (4) 150-362 251 0.307 (0.003) 0.244-0.385

Probable genotype BMI category [Insulin] (SEM) Range Cut point [Glucose] @EM) Range Cut point BMI @EM) Range C.

(N2)

(F2)

(B6D2

ob/+

ob/ob >0.462 (n = 96) 708.1 (42.1) 2.0-1478.0 428(15) 54-776 0.617 (0.008) 0.466-0.831 Fl X B6D2

(F2) (B6D2 ob/+ Flo)

M+)l+) 0.400 (n = 152) 1056.8 (58.5) 33.6-3952.8

W(+)/+) 0.417 (n = 136) 266.6 (29.0) 5.6-1959.2 452 (14) 123-784 0.551 (0.002) 0.422-0.797

Note. BMI = (weight in g)/(nose-to-anus length in cm)2). [Insulin] = pU/ml in plasma. [Glucose] = mg/dl in plasma. [Cut point = mean + 4.265 SEM for parameters in lean (low BMI) animals after preliminary characterization of high BMI and low BMI animals.] For each cross, cutoffs in BMI were selected after inspecting the stem leaf plots in Fig. 3. The plasma glucose and insulin concentrations for the high and low BMI animals are shown. These cut points were used to assess the appropriateness of each animal’s preliminary genotypic assignment based on BMI.

not differ significantly in mean BMI (P -c 0.05 for difference between groups). We also noted that the obese progeny (N2) of the intraspecific backcross displayed a wide range of plasma glucose concentrations (54-776 mg/dl). This is important information relevant to the development of Type II diabetes in these mice. While congenic

MOLECULAR

TABLE Animals

Excluded

ob N2: Possible ob/ob Animals [Insulin] > 4.265 SEM above ID

BMI

Weight

0220 0207 0195 0212 0251 0202

0.385 0.362 0.362 0.353 0.291 0.260

44.9 38.4 41.4 39.0 30.3 23.5

Note. Characteristics further analysis. These insulin concentrations

SE% M M M M M F

MAPPING

OF

2

with BMI < 0.387, Mean for Animals

277.6 199.2 175.8 222.4 200.2 171.2

Analysis and with

[Glucose] 163 232 224 281 248 194

b

Tcrb

collagen

Cpa

bds

1059

ob MUTATION

S

bsl

b

23

s

4

s

6

[Glucose] +/or BMI -C 0.387 Disposition Not scored Not scored Not scored Not scored Not scored Not scored

of six animals which were excluded from were low BMI animals with high glucose or as defined by the cut points in Table 1.

C57BL/GJ ob/ob and db/db animals are insulin resistant and generally euglycemic, congenic DBA mice carrying obesity mutations develop islet cell decompensation and generally become severely diabetic (Hummel et al,, 1972; Leiter et a.Z., 1981; Coleman and Hummel, 1975). If the difference between C57BL/6J and DBA/BJ mice regarding the development of secondary diabetes were a consequence of a single dominant gene, we would have seen a bimodal distribution of plasma glucose concentrations among the obese N2 animals. The absence of bimodality in the distributions for plasma concentrations of insulin and glucose among the ob/ob animals in the backcross (see Fig. 3; note the near continuous distribution of glucose among the high BMI N2 animals) suggests that more than one unlinked allelic difference between C57BL/ 6J and DBA/BJ is responsible for variability in the development of diabetes. 2. RFLP

MOUSE

Met

from Genetic

[Insulin]

THE

Mapping

The map position of ob was determined by typing the F2 and N2 animals for RFLPs at several loci on mouse chromosome 6. Four RFLPs were used in this study: Tcrb (T cell receptor 8) (Snodgrass et al., 1985), Cpu (carboxypeptidase A) (Quint0 et al., 1982), Met (an oncogene) (Dean et uZ., 1987), and Cola-2 (a2 procollagen) (Liau et al., 1985). A polymorphism in Tcrb was present within the original spretus population but both alleles were distinguishable from C57BL/6J. This polymorphism within the spretus population is shown in lane 6 of the Tcrb panel in Fig. 4. Met was polymorphic among C57BL/6J, DBA/BJ, and Mus spretus using BglII. Cpu (BglII), Tcrb (TqI), and Cola-2 (ToqI) were only polymorphic between spretus and C57BL,&J. The Southern blots for these RFLPs are shown in Fig. 4. Because BXD polymorphisms are not yet available for CPU, Tcrb, and Cola-2, initial mapping was per-

FIG. 4. RFLPs used for genetic mapping. Southern blots with DNA from C57BL/6J (b) and Mus spretus mice (s) are shown for a2 procollagen (!!‘a& carboxypeptidase A (BglII), and T cell receptor j3 (TaqI). Polymorphisms for the oncogene Met (BglII) also using b, s, and DBA/SJ DNA (d) are shown. In the a2 procollagen panel, lanes 1, 2, 3 refer to three different 05 spretus progeny. In the Tub panel, 4,5,6 are DNAs from three different progeny.

formed on the progeny of the spretus cross. These probes were mapped among the obese (ob/ob) interspecific F2 animals, and the pedigree analysis is shown in Fig. 5. No double crossovers were noted across the interval between Cola-2 and Tcrb. These data position ob between Tcrb and Met (Fig. 6). The position of three loci on mouse chromosome 6 compares well with the consensus map of this chromosome with the exception of Cola-2. It should be noted that the previous positioning of Cola-2 on proximal chromosome 6 was made using a two-point cross with Met (Irving et al., 1989). The precise position of Cola2 relative to Met was not determined in this study. Of note, there were no recombinants between Cpa and ob among 123 meioses. This places ob between 0 and 2.3 CM from Cpu with 95% confidence. Since these markers all map to human chromosome 7q, these data suggest that the human homologue of ob is likely to map to 7q 31-qter (Fig. 6) (Nadeau, 1989).

n H q q tlB00

= procollagen Met

Cl

ob

clclnoo

Cpa Tcrb

cloclmn

Cl

q ooma 102

1

5

0

15

FIG. 6. Pedigree analysis. The number of animals from the interspecific cross with each DNA haplotype for the RFLPs listed above is shown. Open boxes are C57BL/6J allele; solid boxes represent the spretus allele.

1060

FRIEDMAN Cd Ill

Met

(7q31) 5

04 cpa (7q31) I I

Tcrb 12

ET

mouse ob mutation. To ensure correct assignment of genotype at the ob locus we have measured body mass index (BMI = Wt/length’) and concentrations of plasma glucose and insulin in all the F2 and N2 progeny. This protocol, which makes primary use of BMI and secondary use of plasma concentrations of glucose and insulin, separates ob/ob and wild type into two nonoverlapping groups (data not shown). While these experiments are ultimately aimed at the cloning of the mouse ob gene, several aspects of the data are of general interest. First, we have had the opportunity to characterize the ob phenotype among the progeny of matings that differ in the degree of outcrossing. To diminish the possibility of misassigning the ob genotype, we used measures of plasma concentrations of glucose and insulin in conjunction with BMI to ensure unambiguous assignment of genotype at the ob locus. We noted more overlap between the obese and nonobese groups (as defined by BMI, and concentrations of glucose and insulin) in the interspecific intercross than in the two intraspecific crosses (see Fig. 3). Differences in the phenotypic characteristics of F2 from the interspecific versus the intraspecific intercross reflect the contribution of spretus alleles in the Fl female. By comparing F2 animals from the two crosses, we found that the greater overlap between lean and obese interspecific F2 animals is not due to increased BMI of the lean progeny (as would be expected from typical hybrid vigor), but rather to decreased BMI of the obese animals. Thus, one or more spretw alleles apparently diminish the penetrance of ob with regard to adiposity. This effect appears to be dominant since only one spretus allele is transmitted. This property of Mm spretus may be a consequence of the fact that this strain is not very many generations removed from living in the wild. In addition, we noted wide ranges of plasma concentrations of glucose and insulin among all the obese

(7q32-36) I

FIG. 6. ‘Genetic map of ob on mouse chromosome 6. The map position of 05 relative to (~2 procollagen (Cola-Z), Met, carboxypeptidase A, and T cell receptor 6 (Tcrb) is shown. The map position of each of these genes in the human is shown in parentheses.

Since Met was also polymorphic with DBA, it was mapped among the progeny of all three crosses. In the spretus cross, the male meiosis (C57BL/6J vs DBA/ 25) can be scored independently of the female meiosis (C57BL6J vs spretus) because the alleles for met differ among the three strains. The distance between Met and ob is shown in Table 3 as a function of both the cross and the sex of the gamete. In the intraspecific backcross, only male meiotic events are scored and in the intrasprecific intercross, the genetic distance represents an average of male and female meioses. While there is a tendency to diminish map distance in the interspecific cross, this difference did not reach statistical significance. Such a decrease in map distance might be expected if there were crossover suppression in the interspecific cross to Mus spretus (Robert et al., 1985). There was, however, evidence for increased recombination in the DBA intercross versus the backcross. This is likely a consequence of the fact that the intercross records female and male meiotic events while the backcross records only male meiotic events. Rates of recombination in females are generally higher than those in males except at the telomere. Since ob is near the centromere this point may not be relevant to these crosses (Davisson and Roderick, 1981; Donis-Keller et al., 1987). DISCUSSION

In this report, we describe the mapping of molecular markers in three genetic crosses segregating the TABLE Strain Cross N2 F2 F2 F2

and Sex Effects

3

on Recombination

Fraction

Pl

Male (DBA) Male (DBA) + femaIe Female (spretus) Male

(DBA)

(Met

vs ob)

Recombination fraction

Sex and strain of meioses scored

Strains B6D2 ob/+ Fl X B6 ob/ob female B6D2 ob/+ x B6D2 ob/+ B6D2 ob/+ Fl male X B6 spretus ob/+ Fl female B6D2 ob/+ Fl male X B6 spretus ob ob/+ Fl female

AL.

(DBA)

95% Confidence interval

4/193 22/294 5/127

= 0.021 = 0.075* = 0.039

0.001-0.041 0.045-0.105 0.006-0.073

5/123

= 0.041

0.006-0.076

Note. Since met has different RFLPs among all three progenitor strains (C57BL/6J; DBA/PJ, spretus), it was mapped on the progeny of all three crosses (Fig. 1). Backcross data reflect only intraspecific male meioses and the intraspecific intercross reflects an average of male and female intraspecific meiotic events. In the interspecific intercross, interspecific female meioses could be distinguished from intraspecific male meioses since met had three distinguishable alleles (Fig. 5). * P = 0.054 for difference from recombination fraction for N2.

MOLECULAR

MAPPING

OF THE

F2 and N2 progeny. These ranges of glucose and insulin concentrations exceeded those generally seen in congenic C57BL/6J ob/ob mice (Hummel et al., 1972; Coleman and Hummel, 1975; Leiter, 1981; Leiter et al., 1981). These data are consistent with the idea that differences between inbred mice lines in the development of secondary diabetes in ob/ob animals are a result of polygenic inheritance. Similar conclusions regarding the genetics of diabetes have been reached in other circumstances as well (Bahary et al., 1990; Kaku et at., 1988, 1989). We have generated a map of ob relative to four markers on mouse chromosome 6 by using 123 obese F2 progeny of an interspecific intercross (Tcrb, CPU, Met, and Cola-2) and 200 obese and lean N2 progeny of an interspecific backcross (Met). These data position ob 11.8 CM proximal to Tcrb and 2-4 CM distal to Met. Of note, 0 recombinants of 123 storable animals were noted between ob and Cpa in this cross. This suggeststhat Cpa is ~2.3 CM from ob (with 95% confidence). Since Met and Tcrb, which flank ob, map to human chromosome 7q31-34, these data suggest that if there is a human homologue for ob, it is likely to map to this region. This information is currently being used to determine whether RFLPs from this region cosegregate with an obese phenotype in human pedigrees. The availability of a total of 774 meioses segregating ob will allow us to fine map these genes. Any RFLP that is nonrecombinant among all of these animals is likely to be ~0.6 CM from ob (P c 0.05). Thus, the use of these crosses may make it possible to narrow the region where the ob gene resides to less than 1 Mb. To identify additional probes from this region of the mouse genome we have been making use of libraries derived both by chromosomal microdissection and flow sorting of a mouse 4:6 Robertsonian chromosome (N. Bahary, R. Blank, K. A. Albright, S. Cram, J. Pachter, R. L. Leibel, J. M. Friedman, manuscript in preparation; N. Bahary, D. Siegel, R. L. Leibel, J. M. Friedman, manuscript in preparation). It is important to map Cpa among all of these animals. While no BXD polymorphism has yet been detected with CPU, we have selected genomic clones for this gene and are currently searching for BXD molecular size polymorphisms. We have recently identified two CT repeat sequences from this genomic clone and are testing these minisatellite repeat sequences for length variation between C57BL/6J and DBA/BJ (Weber and May, 1989). Alternatively, denaturing gradient gels can be utilized to reveal polymorphisms. We have been able to map Met among all of the animals since a BXD polymorphism is available for this probe. There is a tendency for more Met vs ob recombination events in the intraspecific intercross than in the intraspecific backcross. This increase may

MOUSE

ob MUTATION

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reflect the fact that the intraspecific intercross summates male and female meioses while the backcross only scores male meioses. It has been previously demonstrated that recombination rates are generally higher in females than in males (Davisson and Roderick, 1981; Donis-Keller et al., 1987). Despite this tendency, the map position of ob relative to Met is not dramatically different in the interspecific cross, suggesting that there is no obvious recombination suppression between Mus spretus and C57BL/6J in this region of the genome. In summary, we have established three crossessegregating the mouse ob gene. We have mapped ob relative to several probes from chromosome 6: Cpa, Tcrb, Met, and Cola-2. This genetic resource should be quite useful in the cloning of the ob gene. The mapping data also position ob in the middle of a homologous group between human 7q22-qter and proximal mouse 6, suggesting that the human homologue of ob, if it exists, is likely to map to 7q31-34. ACKNOWLEDGMENTS We thank L. Cousseau for her expert assistance in preparing the manuscript. We also thank D. Corow for performing ovarian transplants and X. F. Pi-Sunnyer and Y. Dam for performance of the assays for plasma glucose and insulin. This work was supported by National Institutes of Health Grants DK41096, HG00316, and 2P30DK2668. J.M.F. is an Assistant Investigator of the Howard Hughes Medical Institute.

REFERENCES 1. AMAR, L. C., ARNAUD, D., CAMBROU, J., GUENET, J. L., AND AVNER, P. R. (1985). Mapping of the mouse X chromosome using random genomic probes and an interspecific mouse cross. EMBO J. 4: 3695-3700. 2. AVENER, P., AMAR, L., DANDOLO, L., AND GUENET, J. L. (1982). Genetic analysis of the mouse using interspecific crosses. Trends Genet. 4: 18-23. 3. BAHARY, N., LEIBEL, R. L., JOSEPH, L., AND FRIEDMAN, J. M. (1990). Molecular mapping of the mouse db mutation. Proc. Natl. Acad. Sci. USA 8’7: 8642-8646. 4. COLEMAN, D. L. (1978). Obese and Diabetes: Two mutant genes causing diabetes-obesity syndromes in mice. Diubetologia. 14: 141-148. 5. COLEMAN, D. L. (1982). Diabetes-obesity syndromes in mice. Diabetes (Suppl. 1) 31: l-6. 6. COLEMAN, D. L., AND HUMMEL, K. P. (1975). Influence of genetic background on the expression of mutations at the diabetes locus in the mouse. II. Studies on background modifiers. Israel J. Med. Sci. 11: 708-713. 7. DAVISSON, M. T., AND RODERICK, T. H. (1981). Recombination percentages. “Genetic Variants and Strains of the Laboratory Mouse,” Fisher Verlag, Stuttgart. 8. DEAN, M., KOZAK, C., ROBBINS, J., CALLAHAN, R., O’BRINE, S., AND VANDE WOUDE, G. F. (1987). Chromosomal localisation of the met protooncogene in the mouse and cat genome. Genomics 1: 167-173. 9. DONIS-KELLER, H., GREEN, P., HELMS, C., CARTINHOUR, S.,

1062

10. 11. 12.

13. 14.

15. 16.

17.

18.

FRIEDMAN

WEIFFENBACH, B., STEPHENS, K., KEITH, T. P., BOWDEN, D. W., SMITH, D. R., LANDER, E. S., BOTSTEIN, D., AKOTS, G., REDIKER, K. S., GRAVIOUS, T., BROWN, V. A., RISING, M. B., PARKER, C., POWERS, J. A., WAN, D. E., KAUFFMAN, E. R., BRICKER, A., PHIPPS, P., MULLER-KAHLE, H., FULTON, T. R., NG, S., SCHUMM, J. W., BFUMAN, J. C., KNOWLTON, R. G., BARKER, D. F., CROOKS, S. M., LINCOLN, S. E., DALY, M. J., AND ABRAHAMSON, J. (1987). A genetic linkage map of the human genome. Cell 51: 319-337.FEINBERG, A. P., AND VOGELSTEIN, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13. HERBERT, V., LAU, K. S., W., G. C., AND BLEICHER, S. J. (1965). Coated charcoal immunoassay of insulin. J. Clin. Enclocrinol. Metab. 25: 1375-1384. HUMMEL, K. P., COLEMAN, D. L., AND LANE, P. W. (1972). The influence of genetic background on expression of mutations at the diabetes locus in the mouse. I. C57BL/KsJ and C57BL/6J strains. B&hem. Genet. 7: l-3. INGALLS, A. M., DICKIE, M. M., AND SNELL, G. D. (1950). Obese, a new mutation in the house mouse. J. Hered. 41: 317-318. IRVING, N. G., HARDY, J. A., BAHARY, N., FRIEDMAN, J. M., AND BROWN, S. D. M. (1989). The o2 chain of type 1 collagen does not map to mouse chromosome 16 but maps close to the met proto-oncogene on mouse chromosome 6. Cytogener. Cell Genet. 60: 121-122. JOHNSON, P. R., AND HIRSCH, J. (1972). Cellularity of adipose tissue depots in six strains of genetically obese mice. J. Lipid. Res. 13: 2-11. KADISH, A. H., Lrrr.~~, R. L., AND STEINBERG, J. C. (1968). A new and rapid method for the determination of glucose by measurement of rate of oxygen consumption. Clin. Chem. 14: 116. KAKU, K., FIEDOREK, F. T., PROVINCE, M., AND PERMU?T, M. A. (1988). Genetic analysis of glucose tolerance in inbred mouse strain: Evidence for polygenic control. Diabetes .37: 707-713. KAKU, K., PROVINCE, M., AND PERMUIT, M. A. (1989). Genetic analysis of obesity-induced diabetes associated with a limited capacity to synthesize insulin in C57BL/Ks mice: Evidence for polygenic control. Diabetologia 32: 636-643.

ET AL. 19. KEYS, A., FIDAMZA, F., AND KARVONEN, M. J. (1972). Indices of relative weight and obesity. J. Chronic Dis. 25: 329-343. 20. LEIBEL, R. L., BAHARY, N., AND FRIEDMAN, J. M. (1990). Genetic variation and nutrition in obesity: Approaches to the molecular genetics of obesity. “Genetic Variation and Nutrition,” pp. 90-101, Karger, Basel. 21. LEITER, E. H. (1981). The influence of genetic background on expression of mutations at the diabetes locus in the mouse. IV. Male lethal syndrome in CBA/Lt mice. Diabetes 30: 1034-1044. 22. LEITER, E. H., COLEMAN, D. L., AND HURL, K. P. (1981). The influence of genetic background on the expression of mutations at the diabetes locus in the mouse. III. Effect of H-2 haplotype and sex. Dinbetes 30: 1029-1035. 23. LIAU, G., YAMANDA, Y., AND CROMBRUGGHE, B. D. (1985). Coordinate regulation of the levels of type III and type I collagen mRNA in most but not all mouse fibroblasts. J. Biol. Chem. 260: 531-536. 24. NADEAU, J. J. (1989). Maps of linkage and synteny homologies between mouse and man. Trends Genet. 6: 82-86. 25. QUINTO, C., QUIROGA, M., SWAIN, W. F., W. C. N~ovrrs, J., STANDING, D. N., PICTET, R. L., VALENZUEU, P., AND RUTTER, W. J. (1982). Rat preprocarboxypeptidase A: cDNA sequence and preliminary characterization of the gene. Proc. Natl. Acad. Sci. USA 79: 31-35. 26. RIORDAN, J. R., ROMMENS, J. M., -REM, B., ALON, N., RozMAHEL, R., GRZELCZAK, Z., ZIELENSKI, J., LOK, S., PLAVSIC, N., CHOU, J., DRUMM, M. L., IANNUZZI, M. C., COLLINS, F. S., AND Tsur, L. (1989). Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA. Science 246: 1066-1073. 27. ROBERT, B., BARTON, P., MINTY, A., DAUEIAS, P., WEYDERT, A., BONHOMME, F., CAT-N, J., CHAZ~, D., Gmm, J.-L., AND BUCKINGHAM, M. (1985). Investigation of genetic linkage between myosin and actin genes using an interspecific mouse backcross. Nature 314: 181-183. 28. SNODGRASS, H. R., KISIELOW, P., KIEFER, M., STEINMEIZ, M., AND BEOHMER, H. V. (1985). Ontogeny of the T-cell antigen receptor within the thymus. Nature 313: 592-595. 29. WEBER, J. L., AND MAY, P. E. (1989). Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am. J. Hum. Gene% 44: 388-3961