Biochemical Genetics, Vol. 27, Nos. 1/2, 1989

Evolutionary Relationships Between Laboratory Miceand Subspecies of Mus musculus Based on the
Restriction Fragment Length Variants of the
Chymotrypsin Gene at the Prt-2 Locus
Tomomasa Watanabe, 1'2 Nobumoto Miyashita, 3 Masahiko Nishimura, 4
Naruya Saitou, 5 Yukimasa Hayashi, 6 and Kazuo Moriwaki 3

Received 18 Feb. 1987---Final13 Sept. 1988

Restriction endonuclease fragment length variants in mice were compared bySouthern blot analysis using the cDNA probe pcXP33 for the chymotrypsin
gene. The variants were detected in the restriction patterns generated byfragments from digestions with BgllI, E c o R I , HindlII, Pst/, S a c L and X b a L
The set of protein phenotypes and the restriction patterns o f chymotrypsin
gene were examined in many laboratory strains and wild subspecies. Most
laboratory strains (26 strains) are grouped into a set defined as Set 1, but
only a few laboratory strains (AU/SsJ and five BALB/c sublines) areclassified as belonging to Set 2. Of wild subspecies, only BRV-MPL (M.
brevirostris) can be placed in Set 1, while DOM-BLG and SK/Cam (M.
domesticus) belong in Set 2. The assignment of an appropriate set defined by
the characteristics of the chymotrypsin gene has also been investigated in M.
musculus, two Chinese subspecies, M. y a m a s h i n a i , M. molossinus, and M.

Department of Biochemistry, Institute for Developmental Research, Aichi Prefecture Colony,
Kamiyacho, Kasugai, Aichi 480-03, Japan.
2To whom correspondence should be addressed.
3Department of Cytogenetics, National Insititute of Genetics, Yata, Mishima, Shizuoka 411,
Japan.
¢.
Institute for Experimental Animals, Hamamatsu University School of Medicine, Handacho,
Hamamatsu 431-31, Japan.
Department of Anthropology, Faculty of Science, Tokyo University, Hongo, Bunkyoku, Tokyo
113, Japan.
6Department of Genetics, Institute for Developmental Research, Aichi Prefecture Colony,
Kamiyacho, Kasugai, Aichi 480-03, Japan.
1190006-2928/89/0200-0119506.00/0 © 1989 Plenum Publishing Corporation

120

Watanabe et al.

castaneus, and the evolutionary relationship between laboratory mice andvarious subspecies of Mus has been examined.
KEYWORDS:mice;subspecies;chymotrypsin;restrictionfragmentlengthvariants.
INTRODUCTION
Tryspin and chymotrypsin are digestive proteolytic enzymes of the pancreas.
In the mouse, three genetic loci (Prt-l, Prt-3, and Prt-6) have been reported
for trypsin and one locus (Prt-2) has been reported for chymotrypsin(Watanabe and Tomita, 1974; Watanabe et al., 1976a, 1987). On the basis ofanalyses of the constitution of the genes at the Prt-1 and Prt-3 loci in various
subspecies of Mus musculus, it was suggested that the ancestors of laboratory
strains were probably wild mice from western European, related to M. m.
brevirostris rather than to M. m. domesticus (Watanabe et al., 1987).
However, M. brevirostris may be only a regional group of domesticus, since
Ferris et al. (1983) and Sage et al. (1986) have demonstrated that neither
data from proteins nor data from mtDNA provide any genetic support for
distinction between M. brevirostris and M. domesticus. The Prt-2 locus islocated on chromosome 8 (Watanabe et al., 1976b), and three alleles, Prt-2 a,Prt-2 b, and Prt-2 c, have been described at the protein level (Watanabe et al.,1987). Prt-2 ~ has been found in all laboratory strains and also in mostsubspecies of Mus musculus. In contrast, Prt-2 b has been identified only inJapanese wild mice, M. m. molossinus, and in Korean wild mice, M. m.
Table L Listof LaboratoryStrains and the CountriesWhereTheyAre Established

StrainLaboratoryinbred
A/J, AKR/FuA,AU/SsJ, BALB/cAn,
BALB/cByA,BALB/cCdA,BALB/cHeA,
BALB/cRglA,CBA/ByA,CE/JA,
C3H/He, C57BL/6J,C57BL/6ByA,C58/JA,
DBA/2, and 129/SvJ
DD/HeAf, DS, and TSI/A
CFO, MAS/A, and SJL/JA
020/A
RIII/SeA
WLL/BrAf
NZB
KK, N, NC, and SS
Laboratoryclosedcolony
ICR
DDY

SourceUnited States
WestGermany
Switzerland
The Netherlands
FranceNorwayNew ZealandJapan
United States
West Germany

121

ChymotrypsinRestriction Fragment Length Variants in Mice
Table II. List and Sources of the Subspecies of Wild Mice Examineda

Subspecies and stock designation

Site of original collection

Mus m. domesticus

DOM-PGN
DOM-BLG
SK/CamPigeon, Canada
Pomorie, Bulgaria
Skokholm, United Kingdom

Mus m. brevirostris

BRV-MPL

Montpellier, France~Mus m. musculus

MUS-BLG
MUS-NJL

Vrania, Bulgaria
Northern Jutland, Denmark

Chinese subspecies (unidentified)
Sub-BJN
Sub-SHH

Beijing, China
Shanghai, China

Mus m. yamashinai

Sub-ACI
Sub-LAS
Sub-KJR

Suwon, Korea
Suwon, Korea
Kojuri, Korea

Mus m. molossinus

MOL-NEM
MOL-ANJ
MOL-MOM
MOL-WAK
MOL-TSU

Nemuro, Japan
Anjo, Japan
Nagoya, Japan
Wakayama, Japan
Tsushima, JapanMus m. castaneus

CAS/J
CAS-QZN
CAS-TCH

ThailandQuezon City, Philippines
Taichung, Taiwan"Taxonomicassignment is accordingto the reports by Yonekawa et al. (1982) and Suzuki et al.
(1986).

while P r t - 2 ~ was detected in another species, M u s caroli, fromThailand.
In the present paper, we describe our analysis of the restriction fragment
length variants in mice using the recombinant plasmid D N A (pcXP33) thatcarries rat chymotrypsin c D N A inserts as a probe (Bell e t al., 1984) and we
discuss the evolutionary relationships between strains of laboratory mice andvarious subspecies of M u s m u s c u l u s .

yamashinai,

MATERIALS AND METHODS
a n d C h e m i c a l s . The mice used are listed in Tables I and II.
Restriction endonucleases, B g l l I , E c o R I , H i n d l I I , P s t I , S a c I , and X b a I , were

Animals122

Watanabe et aL

purchased from Toyobo Biochemicals. Proteinase K was obtained from
Boerhinger-Mannheim Co., and nylon membrane was purchased from Pall
Ultrafine Filtration Co. Two kinds of agarose, GP-36 and EPI-56, which wereused for the electrophoretic separation of proteins and DNA respectively,
were purchased from Nakarai Chemical Ltd. [a-3ZP]dCTP was obtained fromICN Radiochemicals, and the nick-translation kit was from Amersham.
Other reagents were commercial preparations of the highest purity available.

Gel Electrophoresis for Examination of Bands with Proteolytic Activity. Agarose gel electrophoresis of extracts from the pancreas and duodenum
and the detection of proteolytic activity in bands have been described
previously (Watanabe and Tomita, 1974; Watanabe et al., 1976a).
Preparation of DNA. DNA was isolated from liver, kidney, or lung.
Tissue was homogenized in 10 vol of buffer that contained 20 mM Tris-HCl at
pH 8.0, 10 mM KC1, and 0.25 M sucrose. The homogenate was centrifuged at
300g for 15 min at 4°C, and the pellet was dissolved in 4 vol of 50 mM
Tris-HC1 (pH 8.0), 10 mM EDTA, and 1.25% sodium dodecyl sulfate (SDS).
The solution was then incubated with proteinase K at a final concentration of
0.2 mg/ml at 45°C overnight. Nucleic acid was twice extracted from the
mixture by the addition of an equal volume of phenol-chloroform-isoamyl
alcohol (50:50:1) and was precipitated with 2 vol of ethanol at -20°C
overnight. The pellet was dissolved in a buffer that contained 10 mM Tris-HC1(pH 8.0) and 1 mM EDTA.
Digestion with Restriction Endonucleases. Ten micrograms of DNA
was digested in a total volume of 30 /~1 of reaction buffer with 20 U ofrestriction enzyme at 37°C for 2 hr.
Analysis by DNA Blotting. Electrophoresis on 0.9% agarose gels was
carried out on slab gels (0.5 × 12 × 14 cm) in 0.09 M Tris, 0.09 M boric acid,and 2.5 mM EDTA at pH 8.5 for 4 hr at 75 V. After electrophoresis each gel
was stained with 0.1 ~g/ml ethidium bromide. Transfer of DNA fragments toa nylon membrane was performed as described by Southern (1975). The
chymotrypsin gene from the rat has been cloned and sequenced by Bell et al.
(1984). The plasmid DNA (pcXP33) was nick-translated in the presence of[ot-32p]dCTP (Rigby et al., 1977) to give a specific activity of 1-3 × 108
cpm//zg. Hybridization was performed essentially as described by Wahl et al.
(1979) except that the hybridization mixture contained 50% formamide anddid not contain dextran sulfate. Prehybridization was carried out for 4-5 hr at
42°C and hybridization was allowed to take place for 20 hr at 42°C.
Hybridizing fragments were visualized by autoradiography with Kodak
XAR-5 film.

ChymotrypsinRestriction Fragment Length Variants in Mice123

RESULTSThe restriction endonuclease fragment length variants in mice were clearly
detectable by Southern blot analysis using the chymotrypsin cDNA probe
pcXP33, which contains 370 base pairs of the open reading frame corresponding to amino acids 33 to 156. The variants were detected in the
restriction patterns generated by digestions with BgllI, EcoRI, HindlII, PstI,
SacI, and XbaI, whereas in the case of digests with BamHI, EcoRV, and KpnI
no difference in restriction patterns was observed in any mice examined.

Bg.!II

HindllIC C CC BB C AAABBCCBAAAA
3riginOrigin

?.31
94
6.6
4.4

-23.1
- 9.4
- 6.6
- 4/,

23

- 2.3
" 2D
kb

2.0
kb

Xbal

Pst I
8BBBBCBAAAA BB AA

A AA

AA"3rigin

Origin

.~3.1
9.4
6.6
4.4

23.1
9.4
6.6
4.4

23
2.0
kb

23
20
kb

Fig. 1. The restriction endonuclease fragment length variants of the chymotrypsin gene frommice examined by Southern blot analysis using a rat chymotrypsin cDNA pcXP33 probe.

Watanabe et aL

124

Table IlL Restriction Fragment Length Variants of the Chymotrypsin Gene from the Mouse
Fragment length (kb)
Restriction enzyme

A

B

C

BgllI

7.0

8.0

6.0

EcoRI

7.6
7.3

8.3
7.3

HindIII

5.8
2.3

4.2
3.3

5.8
3.3

PstI

3.0
2.4

3.6
2.4

3.6
2.0

SacI

11.0

4.2

XbaI

9.3

4.3

Examples of restriction patterns and a summary of the different types ofpattern are presented in Fig. 1 and Table III, respectively. Three patterns (A,B, and C) were observed in the case of fragments produced by BgllI, HindllI,and PstI, while two patterns (A and B) were obtained when EcoRI, SacI, and
XbaI were used. Although there are two types of protein variants showing
different electrophoretic mobilities, the strain distributions in the phenotypeof the protein were not consistent with those of any types of the restriction
pattern (Table IV), indicating that the variable site in the protein Variants isdifferent from the polymorphic sites detected in the DNA variants. Genomic
DNA encoding rat chymotrypsin has been isolated and sequenced (Bell et al.,Table IV. The Set of Protein Phenotypes and Types of Restriction Fragment Length Variantsof the Chymotrypsin Gene from Subspecies of Wild Mice
Restriction type
StrainProteinSet phenotype BgllI

EcoRI HindlII PstI SacI XbaIBRV-MPL and DOM-PGN

1

A

A

A

A

A

A

A

DOM-BLG and SK/Cam

2

A

C

B

C

B

B

B

MUS-BLG, MUS-NJL,
sub-BJN, and sub-SHH

3

A

B

B

B

B

B

A

Sub-ACI, sub-LAS, sub-K JR,
MOL-NEM, MOL-ANJ,
MOL-MOM, and
MOL-WAK

4

B

B

B

B

B

B

A

MOL-TSU, CAS/J,
CAS-QZN, and CAS-TCH

5

A

B

B

B

C

B

A

ChymotrypsinRestriction Fragment Length Variants in Mice

1251984), but that from the mouse has not. At the present time, as we did notconstruct the restriction maps of mouse chymotrypsin genomic DNA, it
remains to be determined whether the polymorphic sites are in coding or
noncoding regions.
Since all of several polymorphic sites are involved in one chymotrypsin
genome, their variants behave as a set of genetic elements in the mouse
populations. In order to analyze the genetic relationships between mice, theset of protein phenotypes (Watanabe et al., 1987) and the types of restriction
fragment variants of the chymotrypsin genomic gene were examined for manylaboratory strains and wild subspecies of mice (Tables IV and V). Most of thelaboratory strains can be grouped into Set 1, which includes the following
characteristics: A in protein phenotype, A type in BgllI, A in EcoRI, A in
HindlII, A in PstI, A in SacI, and A in XbaI. In contrast, only the AU/SsJ
strain and five BALB/c sublines (BALB/cAn, BALB/cByA, BALB/cCdA,
BALB/cHeA, and BALB/cRglA) were classified as belonging to Set 2, being
A, C, B, C, B, B, and B, respectively. Of European wild subspecies,
BRV-MPL (M. brevirostris), as well as DOM-PGN (a strain of M. domesticus from Canada), could be placed unequivocally in Set 1 with most of thelaboratory strains. DOM-BLG and SK/Cam, which are M. domesticus from
Bulgaria and the United Kingdom, resepectively, fell into Set 2 with AU/SsJ
and the five BALB/c sublines. Two M. musculus strains of the MUS-BLG
and MUS-NJL subspecies were classified as belonging to Set 3, being A, B, B,
B, B, B, and A. As the populations of Asian wild mice have sometimes beenshown to possess unique genetic characters different from those of European
wild subspecies and laboratory mice, we chose several strains representative of
Asian wild subspecies and examined the restriction fragment length variantsof their chymotrypsin genes. The Chinese subspecies clearly belonged to Set 3,
as did M. musculus. The Korean wild subspecies (M. yamashinai) and fourJapanese wild strains (M. molossinus) belonged to a fourth set, Set 4, having
Table V. The Set of Protein Phenotypes and Types of Restriction Fragment Length
Polymorphismof the Chymotrypsin Gene in Laboratory Mouse Strainsi

Restriction type
StrainSet

Proteinphenotype BgllI

EcoRI

HindlII

PstI

SacI

XbaI

26 laboratory strainsa

1

A

A

A

A

A

A

A

6 laboratory strainsb

2

A

C

B

C

B

B

B

i

aA/J, AKR/FuA, CBA/ByA, CE/JA, C3H/He, C57BL/6J, C57BL/6ByA, C58/JA, DBA/2,
129/SvJ, DD/HeAf, DS, TSI/A, CFO, MAS/A, SJL/JA, 020/A, RIlI/SeA, WLL/BrAf,NZB, KK, N, NC, SS, ICR, and DDY.
bAU/SsJ, BALB/cAn, BALB/cByA, BALB/cCdA, BALB/cHeA, and BALB/cRglA.

126

Watanabe et al.

characteristics B, B, B, B, B, B, and A, and a M. molossinus strain,
MOL-TSU, and three strains of M. castaneus from Thailand, the Philippines,
and Taiwan belonged to Set 5, having characteristics A, B, B, B, C, B, andA.
We estimated the number of nucleotide substitutions in the different
fragments of the five sets. One nucleotide substitution was expected between
every pair of types in the three types that generated different BglII restriction
fragments. One nucleotide substitution was also expected between the two
types that generated different E c o R I fragments. In the HindlII digests, one
nucleotide exchange was estimated between the A and the C types and also
between the B and the C types, while two were estimated between the A andthe B types. In the case of PstI fragments, one nucleotide substitution was
estimated between the A and the B types and also between the B and the Ctypes, while two were estimated between the A and the B types. Between the
two types identified by both the SacI and the XbaI digests, one nucleotide
exchange was expected. From these results, genetic divergence among the fivesets was calculated by the procedure of Nei and Li (1979), and thedendrogram was drawn as shown in Fig. 2; in this case, the difference ofprotein phenotype was not taken into account in the degree of genetic distance,
as it is not exactly clear that the different protein phenotypes had arisen froma nucleotide substitution in a site different from any of the restriction sites.
The genetic relationship between the members of Set 1 and those of the otherfour sets is the most distant. Next, Set 2 is more distant from the other threesets. The relationship among Sets 3, 4, and 5 is very close; Sets 3 and 4 seem to
be closest.

Set 1
Set 2
Set 5
Set 3
Set 4
Genetic

i

distance 0

,

,

,

,

,

,

.01

.02

.03

.04

.OSS

.06

Fig. 2. Dendrogram showing the genetic relationship among the five sets ofchymotrypsingenes.

ChymotrypsinRestriction Fragment Length Variants in Mice127

DISCUSSION
Since DNA variants include the differences in sequence that correspond not
only to amino acid substitutions but also to those that cause no exchanges ofamino acids in exons and even in noncoding regions of introns, they are much
more useful for finding genetic variations, as shown, for instance, in serum
amyloid A protcins (Taylor and Rowe, 1984). DNA variation in mice waspreviously reported in mitochondrial DNA (mtDNA), and the reports on
mtDNA variations have demonstrated that strains of laboratory mice were
derived primarily from the European subspecies M u s m. domesticus (Yonekawa et al., 1980, 1982, Ferris et al., 1982, 1983). The M. brevirostris type of
mtDNA has been found only in NZB sublines and CF1 mice (Yonekawa etal., 1982; Wakana and Kato, personal communication), and other laboratory
strains have all been shown to be of the M . domesticus type. Furthermore,
there is evidencc that wild mice from various locales show a high level ofrestriction-site variations of mtDNA associated with their rapid evolution, but
no such variations are found among most laboratory strains (Ferris et al.,
1982, 1983). It has been proposed from these findings that only one female of
M. domesticus contributed to the gencration of most laboratory strains.
However, as Suzuki et al. (1986) have pointed out, the mtDNA variations
may not reflect the true evolutionary relationship between subspecies because
mtDNA is only maternally inherited, and therefore, it is essential to examine
nuclear DNA also. Suzuki et al. (1986) intensively investigated the restriction
fragment length variants of the nontranscribed spacer region with ribosomal
DNA (NTS) from various subspecies of M u s musculus. In their study, theyfound that most laboratory strains examined also possessed the M. domesticus
type of NTS. The fact that some nuclear genes in laboratory mice are derived
from M. m. domesticus is in accord with the results from the mitochondrial
genomes.
Since previous studies of variations in either proteins (Bonhomme et al.,1984) or mtDNA polymorphism (Ferris et al., 1983) failed to detect marked
differences between M. domesticus and M . brevirostris, these subspecies areoften combined as a single taxonomic unit, M. m. domesticus. However,according to the old morphological classification of Schwarz and Schwarz(1943), mtDNA polymorphism (Yonekawa et al., 1982), and heterogeneity ofribosomal DNA (Suzuki et al., 1986), M. domesticus and M. brevirostris do,in fact, have different genetic characteristics. The protein phenotype at thePrt-1 and Prt-3 loci in many strains of laboratory mice coincides not with theM. domesticus phenotype but with that of M. brevirostris (Watanabe et al.,1987). Furthermore, in the present study, the set of protein phenotypes andtypes of restriction fragment length variants of the chymotrypsin gene at thePrt-2 locus in most laboratory strains is consistent with that of M. m.
brevirostris, since they are all grouped into Set 1. Only a M. domesticus strain

128

Watanabe et aLfrom Canada belonged in Set 1, even though Selander and Yang (1969)
indicated that the wild mice in North America, which were introduced from
Europe, might be M. brevirostris. Both strains of M. domesticus from
Bulgaria and the United Kingdom are classified into Set 2 as are AU/SsJ andfive BALB/c sublines. As shown in Tables IV and V, the nucleotide sequencesof the chymotrypsin genes in Sets 1 and 2 are expected to be clearly different.
So many differences in their sequences are not considered to have occurred in
the course of breeding of laboratory strains but are assumed to have resulted
from the variations in wild populations. Therefore, laboratory strains might bemixed as the result of a cross between M. domesticus mice and M. brevirostrismice.
AU/SsJ and five BALB/c sublines are grouped in Set 2. AU/SsJ is wellknown to possess Hbb p (Morton, 1966), which has not been found in western
European wild mice but has been detected in Asian wild mice (Japan, Korea
and China), Polish and Bulgarian wild mice, and wild mice in Israel, Greece,and Italy (Ritte and Neufeld, 1982; Miyashita et al., 1985). In the present
study, it has also been demonstrated that BALB/c sublines have a chymotrypsin gene that differs from that of many other laboratory strains.
Asian wild mice such as the Chinese subspecies, M. yamashinai, M.
rnolossinus, and M. castaneus, appear to be closely related genetically fromthe DNA restriction patterns of their chymotrypsin genes. M. molossinus (aJapanese wild mouse) is divided between Set 4 and Set 5. Since M.
yamashinai and the Chinese subspecies are classified into Set 4 and M.
castaneus is in Set 5, M. molossinus must be derived and mixed from M.
yamashinai or the Chinese subspecies and castaneus. This result is consistent
with the investigations of mtDNA polymorphism (Yonekawa et al., personalcommunication) and the distribution of H b t f (Miyashita et al., 1985) in M.
molossinus captured in several places in Japan. The Chinese wild subspecies
are also divided between Set 3 and Set 4. The set of some Chinese subspecies
was the same as those of two strains of M. musculus from Bulgaria andDenmark, because they are classified as belonging to Set 3. The area
associated with mice in Set 3 is very large, and the wide distribution is similar
to the geographical distribution of heterogeneity of ribosomal DNA (Suzuki
et al., 1986). M. rnusculus seems to function to mediate the gene migration
between European wild mice and Asian wild mice.
The difference in protein phenotypes (A and B) transcribed from thechymotrypsin gene did not coincide entirely with the restriction types of theDNA. For example, as seen in the correlation between Set 1 and Sets 2, 3, and5, even if the protein phenotype is A, the restriction types are different fromone another. Conversely, although the protein phenotypes are different fromeach other in that Set 3 is A and Set 4 is B, their restriction types were all thesame.

Chymotrypsin Restriction Fragment Length Variants in Mice129

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