Evolutionary Implication of Heterogeneity of the Nontranscribed ...

Evolutionary Implication of Heterogeneity of the Nontranscribed Spacer Region of Ribosomal DNA Repeating Units in Various Subspecies of Mus musczdzd Hitoshi Suzuki,* Nobumoto Myashita,* Kazuo Moriwaki,* Ryo Kominami,“f Masami Muramatsu,? Takeharu Kanehisa,$ Franqois Bonhomme,$ Michael L. Petras,”Ze-chang Yu,# and De-yuan Lu** *National Institute of Genetics, Japan; TTokyo University; $Kobe University; &Jniver& des Sciences et Techniques du Languedoc, Montpellier, France; “University of Windsor, Ontario, Canada; #National Vaccine and Serum Institute, Beijing, People’s Republic of China; and **Shanghai Second Medical College, People’s Republic of China

Genetic variability of the nontranscribed spacer (NTS) region within ribosomal DNA repeating units in the various subspecies of Mus musculus was determined. Mice belonging to several laboratory mouse strains were examined by means of Southern blot hybridization with a mouse ribosomal DNA probe. This probe encompasses the 3’end of the 28s ribosomal RNA (rRNA) gene and the following spacer. Restriction enzyme digestions of the liver DNAs from various wild mice revealed that each of the subspecies has a unique pattern in the spacer encompassing a distance - 10 kb downstream from the ribosomal gene. These restriction patterns permit the classification of mouse subspecies and also provide insights into the origin of the laboratory mouse strains. Introduction

Recent studies on the restriction fragment-length polymorphism (RFLP) of mitochondrial DNA (mtDNA) suggested that laboratory mouse strains were derived primarily from the European subspecies Mus musculus domesticus (Yonekawa et al. 1980, 1982; Ferris et al. 1982). However, since mtDNA is maternally inherited (Huchinson et al. 1974) and may cross subspecies boundaries (Ferris et al. 1983a; Boursot et al. 1984), its restriction patterns may not reflect the true evolutionary relationship of subspecies involved. It was therefore considered essential to examine nuclear DNA, specifically ribosomal DNA (rDNA), for such relationships. Variations in rDNA follow Mendelian patterns of inheritance. There are 100-200 copies of mouse rDNA genes in the genome (Atwood et al. 1976). They are tandemly repeated in clusters at several different sites on the chromosomes. Each rDNA repeating unit is composed of a coding region that is transcribed into the precursor molecule for 28S, 5.8S, and 18s rRNA and a nontranscribed spacer (NTS). The NTS in rDNA is known to evolve rapidly in most of the higher eukaryotes (Arnheim 1983), and restriction analysis of such DNA has already been shown to 1. Key words: restriction fragment-length bosomal DNA, wild mouse subspecies.

polymorphism (RFLP), nontranscribed spacer (NTS), n-

Address for correspondence and reprints: Kazuo Moriwaki, National Institute of Genetics, Mishima, Shizuoka-ken, 4 11, Japan. Mol. Biol. Evol. 3(2): 126-137 1986. 0 1986 by The University of Chicago. All rights reserved. 0737-4038/86/0302-3203$02.00

126

Heterogeneity of Nontranscribed Spacer in Ribosomal Genes

127

provide useful phylogenetic data (Wilson et al. 1984). Restriction-site maps of the rRNA gene region including the NTS have been constructed by Carry et al. (1977) and Kominami et al. ( 198 1) for laboratory mice. In this study, the restriction cleavage patterns of the NTS region of rDNA from various subspecies of Mus musculus (wild and laboratory strains) were compared. Geographical distribution of the rDNA haplotypes based on RFLP provides further insight into the origin of laboratory strains. Material and Methods

Mice The animals used in the present study included wild mice (see table 1 for the collecting localities); laboratory mice A/WySnJ, AKR/J, AU/SsJ, C3H/HeJ, C57BL/ 65, C57BL/lOJ, DBA/lJ, DBA/2J, PL/J, SM/J, and 129/SvSlCP (all originally from the Jackson Laboratory); BALB/cAnN and CBA/CaHN (both originally from the National Institutes of Health); RFM/Ms (from the National Institute of Radiological Sciences, Japan); and NZB/San (from the Institute of Medical Science, University of Tokyo). Blot Analysis Nuclear DNAs were prepared from the livers of the mice as described by Maniatis et al. (1982). They were digested with EcoRI and/or BamHI (Takara Biochemicals, Kyoto, Japan) and electrophoresed on 0.6%-0.7% agarose gel at 3V/cm for 8-l 5 h in 40 mM Tris-acetate buffer (pH 7.8) containing 2 mM ethylene diaminetetraacetate and 0.5 pg/ml ethydium bromide. Then, the double-stranded DNA fragments were transferred to a nitrocellulose filter, heated at 80 C for 3 h, and hybridized with a probe of 0.7-kb double-stranded DNA in 6 X SSC as described by Southern (1975). (1 X SSC = 0.15 M NaCl-0.015 M Na citrate solution with pH 7.0.) The probe was prepared from an EcoRI-6.6-kb rDNA fragment cloned by Kominami et al. (1982). It included the HinfI restriction site at the 3’-end region of 28s rDNA and the BamHI site in the NTS downstream of the 3’end. Labeling of the probe was carried out using (a-32P)dCTP (Amersham Searle) and DNA polymerase I (Boehringer-Mannheim) (Maniatis et al. 1982). The specific radioactivity of the probe was l-2 X lo* cpm/pg. The site of the DNA probe within the DNA repeating unit is illustrated schematically in figure 1 (Kominami et al. 198 1). Prior to hybridization, the filter was incubated at 66 C for 2-4 h in 10 ml of the 6 X SSC containing 0.0 1% (w/v) sodium dodecyl sulfate and Denhardt’s solution with denatured salmon-sperm DNA (100 pg/ml). Hybridization was performed at 66 C for 24 h, and the filter was washed twice with 150 ml of 0.1 X SSC for 1 h. Autoradiography was performed on Fuji-RX film (Fuji-Film Co., Japan) for 12-24 h with an intensifying screen. For a more detailed restriction map near the 3’end of 28s rDNA, the EcoRI or BamHI fragments were digested with EcoRV, PvuII, PstI, or Sac1 endonucleases (Takara Biochemicals, Kyoto, Japan). The resulting fragments were hybridized with the DNA probe. Molecular-Weight

Determination

of DNA

As shown in figure 1, molecular weights of the restriction fragments of the rDNA repeating units were determined using 0.6%-0.7% agarose gel and rDNA fragments such as 2.3-kb BamHI and 6.6-kb EcoRI fragments as molecular markers (Kominami et al. 1981).

128

Suzuki, Miyashita, Moriwaki, et al.

61 B2B3B4

1 18s

28s

Bl

B5

-. -.

:, \ \ \ 0.7

l-.

*\

kb probe .*--_

v

*. -.

E\i_I I : I

. E

l

:

I

J3Hinf I&

B5

4

6.6 kb EcoRl

*

zz BamHl FIG. 1.-A restriction map of mouse ribosomal RNA repeating units constructed according to Kominami et al. ( 1981). The 4% rRNA coding region is depicted by the elongated box. The darkened segments of the box labeled 18s and 28s indicate the genes coding for 18s and 28s rRNA, respectively. The 0.7-kb probe used hybridizes with mouse rDNA from the Hi&I site on the 3’-end region of the 28s rRNA gene to the BumHI site (B4) on the following spacer region.

Results

Heterogeneity Involving EcoRI or BamHI Fragments of rDNA Repeating Units from Wild Mice The EcoRI and BamHI digests of the liver DNAs obtained from 55 individuals collected at 3 1 sites throughout the world (table 1) were hybridized with the 0.7-kb rDNA probe. The patterns obtained are shown in figure 2 and summarized schematically in figure 3. The restriction fragments in the region from the 3’end of 28s rRNA gene to the downstream spacer region varied in length in the different subspecies. As seen in figure 3, at least nine different EcoRI bands and seven BamHI bands were observed. These were 5. l- 10.5 and 2.3-30 kb, respectively. Each subspecies seemed to have a characteristic major band, such as 2.3-kb BamHI in A4us musculus domesticus. These restriction patterns suggest that many types of DNA repeating units occur in the wild mouse populations investigated. In this paper each type is designated as an rDNA haplotype. Restriction-Site

Maps of rDNA Haplotypes

Eight rDNA haplotypes were recognized based on the sizes of the EcoRI, BamHI, and EcuRI + BamHI digests (table 2). To construct the restriction maps for these haplotypes, the genomic DNAs from seven individuals (DOM-LBL, SK/Cam, BRVMPL, MUS-BLG3, MOA, CAS-QZN, BAC-LAH) were digested with both EcoRI and one of the following enzymes: BamHI, EcoRV, PvuII, PstI, and SacI. On double digestion with EcoRI and EcoRV, only a single major fragment of 1.1 kb was observed in all the cases (data not shown). This suggested that the EcoRI


129

Table 1 List and Source of Wild Mouse Subspecies Employed

Subspecies and Site of Original Collection Mus Musculus domesticus: 1. Pigeon (Canada) ...........................

2. L. Belanger (Canada) ....................... 3. Skokholm Island (United Kingdom) ........... 4. Pomorie (Bulgaria) ......................... 5. Langadas (Greece) ......................... M. m. 6. M. m. 7.

Stock Designation (N)

DOM-PGN DOM-LBL SK/Cam a DOM-BLGb DOM-GRCb

(2) (2) (2) (1) (1)

BRV-MPL

(2)

BAC-LAH BAC-KAB

(2) (2)

URB-BDW

(2)

CAS-BGR CAS-QZN CAS-TCH

(1) (2) (2)

brevirostris:

Montpellier (France) ........................ bactrianus:

Lahore (Pakistan) .......................... 8. Kabul (Afghanistan) ........................

M. m. urbanus: 9. Bandarawela (Sri Lanka) M. m. castaneus:

....................

10. Bogor (Indonesia) .......................... 11. Quezon City (Philippines) ................... 12. Taichung (Taiwan) ......................... M. m. musculus:

13. Vrania (Bulgaria) .......................... 14. Northern Jutland (Denmark) M. m. molossinus: 15. Nakashibetsu, Hokkaido (Japan)

16. 17. 18. 19. 20. 21. 22. 23.

..............

Morioka, Iwate Prefecture (Japan) ............. Omiya, Saitama Prefecture (Japan) ............ Mishima, Shizuoka Prefecture (Japan) ......... Anjo, Aich Prefecture (Japan) ................ Teine, Hokkaido (Japan) .................... Ohma, Aomori Prefecture (Japan) ............. Hakozaki, Fukuoka Prefecture (Japan) ......... Kagoshima, Kagoshima Prefecture (Japan) ......

Chinese subspecies (UnidentiJied): 24. Changchun (China) ........................

25. 26. 27. 28. 29. 30. 3 1.

Urumuchi (China) ......................... Jiayuguang (China) ......................... Lanzhou (China) ........................... Chengtu (China) ........................... Beijing (China) ............................ Nanjing (China) ........................... Shanghai (China) ..........................

MUS-BLG3 b (2) MUS-NJL (2) MOL-NSB MOL-MRO MOL-HOM MOL-MSM MOA” MOL-TEN2 MOL-OHM MOLHKZ MOL-KAG

(1) (1) (2) (2)

sub-CHC sub-URM sub-JYG sub-LZH sub-CHT sub-BJN 1 sub-NAN sub-SHH

(1) (2) (1) (2) (1) (2)

(3) (2) (2) (1) (2)

(3) (2)

’Inbred strain obtained through Dr. K. Kondo. b Original nomenclatures were DBP for BLG, DGD for GRC, BRV/2 for MPL, and MBV for BLG3. ’ Inbred strain developed by Dr. K. Kondo.

and EcoRV sites are well conserved in the mouse subspecies examined. The 0.7-kb probe hybridized with the region just downstream of the E2 site (see fig. 1). Therefore, the conservative EcoRV site is probably located 1.1 kb downstream of E2. Both single and double digestions placed BarnHI, PvuII, and Sac1 sites upstream of E2. Those sites were also well conserved in all mice investigated. The restriction maps of the eight rDNA haplotypes (rl-r8) are presented schematically in figure 4.


13 1

DOM IBR” 8 9 IO,11 12 0 13 z 14 ? 15-19

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1 I

I I

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PIG. 3.-Types of restriction patterns of rDNA, the nontranscribed spacer region, in various wild mouse subspecies. Numbers in “Place of collection” correspond to those in table 1. DOM = subspecies Mw musculus domesticus, BRV = A4. m. brevirostris, BAC = M. m. bactrianus, URB = M. m. urbanus, CAS = M. m. castaneus, MUS = M. m. musculus, MOL = M. m. molossinus, and subsp. = unidentified Chinese subspecies. The approximate lengths (kb) of restriction fragments are indicated at the top.

molossinus mice collected from seven localities in Japan (nos. 15-21) exhibited a characteristic minor haplotype, r5. Wild mice from Hakozaki (no. 22) in northern Kyushu had r4 and r8 haplotypes. The latter appears specific for bactrianus (nos. 7, Table 2 EcoRI, BumHI, and EcoRI + BumHI Fragment Lengths in the Mouse rDNA Haplotypes FRAGMENT LENGTH (kb) OF rDNA HAPLOTYPES

rl r2 r3 r4 r5 r6 r7 r8

.... .. .. .. . .. ..

.. .. . ..

....

EcoRI

BamHI

EcoRI + BarnHI”

6.6 6.6 5.5 9.0 8.8 9.0 6.5 7.0

6.5 2.3 30 6.5 6.3 6.4 6.4 >30

5.1 0.9 5.5 5.1 4.9 5.0 5.0 7.0

TYPICAL EXAMPLES OBSERVED

SK/Cam, BRV-MPL, DOM-LBL, SK/Cam, BRV-MPL BRV-MPL MUS-BLG3, MOA MOA CAS-QZN CAS-QZN, BAC-LAH CAS-QZN b, BAC-LAH

’ Double digestion with EcoRI and BumHI. b BamHI band in CAS-QZN was faint; length heterogeneity of the NTS BumHI fragment might be assumed: one being 30 kb and the other the longer.

132

rl


03

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5.1

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E

B5

E

11 B3

E2 B4

r2

IO.9 1 11

B3

r3

E2 I 1

B3

E

5.5

i

E2

r4

5.1

E

B5

11

B3

r5

E2 1

83

r6

4.9

B

E

I

A

E2

5.0

E

B

I

11 83

r7

I

r8

83

E2

I

5.0

E2

B

E

E 7.0

11

I d 1

lkb

PIG. 4.-Restriction maps of mouse rDNA responsible for the 28s rRNA using double digestion analysis. E = EcoRI; B = BarnHI; 0 = EcuRV; A = PvuII; A = PstI; and e = SacI. Ribosomal DNA haplotypes r 1, r2, r3, r4, and r8 correspond to the major repeating units of DOM-LBL, SK/Cam, MUS-BLG3 or MOA, BRV-MPL, and BAC-LAH, respectively. The r5 haplotype is a minor repeating unit observed in almost all mice trapped in Japan. Haplotypes r6 and r7 are submajor repeating units observed in CAS-QZN. The approximate lengths (kb) of EcoRI (E2) and BumHI fragments detected by the 0.7-kb probe are shown. The lengths of E2/B5 and E2/B4 have already been determined by Kominami et al. (198 1).

8) and castaneus (nos. 10-12). A wild mouse collected from Kagoshima (no. 23) exhibited r6 and r8, both of which are castaneus type. Wild specimens collected near Nanjing (no. 30) carried r 1, r4, and r8; those from Shanghai (no. 3 1) showed r4 and r7; and, finally, the major haplotypes of urbanus (no. 9) were r6, r7, and r8. Southern Blot Analysis of Laboratory Mice A blot analysis of repeating units on 15 laboratory mouse strains listed in Material and Methods was carried out. Most of the strains showed the domesticus-type NTSthat is, 6.5-kb and 2.3-kb BarnHI fragments corresponding to the haplotypes rl and r2 (see fig. 4). The SM/J strain, however, showed only the 2.3-kb BarnHI band. Moreover, some strains, such as AU/&J, RPM/MS, PL/J, CBA/CaHN, and NZB/San, also possessed minor fragments not found in wild domesticus. BamHI cleavage of CBA/

FIG. 5.-Geographical distribution of mouse rDNA haplotypes. Numbers refer to the collection sites of wild mice listed in table 1. Percentages of each haplotype except rl and r2 were based on the relative concentration of “EcoRI band” corresponding to each haplotype. Percentage of rl and r2 was based on the concentration of the 6.5-kb and 2.3-kb BumHI bands, respectively. Haplotypes containing -&XI in each genome were neglected in this figure. The sites and bands represented by the unshaded portions of the circle are as follows: (4) and (5), rl; (6), r3; (8), “5.1-kb EcoRI band”; (12), “7.5-kb EcoRI band”; (13), “10.5-kb EcoRI band”; (15)-( 19) and (21), r5; (20),r5 and “7.5-kb EcoRI band.”

134


CaHN and NZB/San DNA yielded fragments more than 6.5 kb long. On EcoRI digestion, PL/J showed a 5.5kb minor component(s). In addition, a 7.0-kb minor band was found in Au/SsJ and RFM/Ms strains. Discussion

Genetic Classification of Wild Mice The NTS region involving the area 10 kb downstream from the 3’end of the 28s rRNA gene was found to be highly variable with respect to the distribution of the restriction sites in wild subspecies of A4us musculus. At least 10 different kinds of rDNA haplotypes were recognized in the genomes of subspecies investigated. Restriction maps of eight of these are presented in figure 4. Each subspecies appears to have a characteristic combination of the haplotypes. Previously, Kuehn and Arnheim ( 1983) reported a polymorphism of the NTS region that lies 2 10 bp upstream from the origin of transcription of the rRNA precursor. Since nothing is known about the distribution of this polymorphism, its usefulness in distinguishing between subspecies is uncertain. The patterns of rDNA organization in the seven subspecies studied--domesticus, brevirostris, bactrianus, urbanus, castaneus, muscuhs, and molossinus-suggest that this could provide us with a new set of criteria for their classification. The subspecies studied may be placed into three groups-domesticus, castaneus, and musculususing the NTS haplotypes (r 1-r8) summarized in table 3. This appears consistent with the conclusions previously reached following mtDNA restriction enzyme studies (Yonekawa et al. 1981; Ferris et al. 1983b) and the hemoglobin beta chain allelic frequencies (Miyashita et al. 1985). Subspecies domesticus and brevirostris are combined into the domesticus group because they have rl, r2, and r3; musculus and molossinus are placed in the musculus group because both have r4 and r5; and bactrianus, castaneus, and urbanus are combined into the castaneus group because they have r6, r7, and r8. The above classification does not match perfectly with the three major biochemical groups (Musl, Mus2, and MUSS)suggested by Bonhomme et al. (1984). In Bonhomme et al.‘s classification, castaneus is placed in the musculus group and bactrianus in a distinct group. Which of these classifications is preferable remains to be settled. Differences were also found between brevirostris (e.g., BRV-MPL) and domesticus (e.g., DOM-LBL, SK/Cam, DOM-BLG, and DOM-CRC). For instance, in BRV-

Table 3 Putative Genetic Classification of Mouse Subspecies the Combinations of the rDNA Haplotypes Groups and Subspecies Domesticus: Mus musculus domesticus M. m. brevirostris . . . . . Castaneus: M. m. bactrianus . . . . . . . M. m. urbanus . . . . . . . M, m. castaneus . . . . . . Musculus: M. m. musculus . . . M. m. molossinus . . . . . .

Based on

Combinations of rDNA Haplotypes

rl, r2, r3

r6, r7, r8

r4, r5


135

MPL, in addition to rl and r2, -50% of the haplotypes are r3, which is characteristic of brevirostris. Previous studies using either protein polymorphism (Bonhomme et al. 1978, 1984; Sage 1981; Thaler et al. 1981) or mtDNA (Ferris et al. 1983b) did not detect marked differences between domesticus and brevirostris. They are, therefore, often combined in a single taxonomic unit, M. m. domesticus (biochemical group Mus 1). However, in the old classification of Schwartz and Schwartz ( 1943), domesticus, being dark colored and having a large body and long tail, is considered the northern form, whereas brevirostris, being light colored and white bellied and having a somewhat shorter tail and smaller body weight, is found in more southern climates. These two morphophytes may have a different ecological preference. The rRNA haplotypes may also be used to give some insight into the evolutionary relationship among the subspecies involved. In this regard the r 1, r4, r6, and r7 types, which are generally observed in wild mouse populations, may be especially informative. E2/B fragments of both r 1 and r4 types are 0.1 kb longer than those of r6 and r7. Two explanations for this may be suggested. One is that r 1 and r4 were derived from r6 and r7. Ancestral castaneus, which carried r6 and r7, moved west and in differentiating to bactrianus lost r6. Subsequently, in domesticus, the rl was derived from r7 through the insertion of a 0. 1-kb DNA in the E2/B fragment. In ancestral castaneus, which differentiated to musculus in the east or north, r6 became r4 by insertion of a 0. 1-kb DNA. A second hypothesis involves the mixing of domesticus-like and musculus-like mice in ancient time. Thereafter, E2/B fragments of rl and r4 were shortened by -0.1 kb in the genomes of the castaneus ancestors. This gave rise to r6 and r7. Such changes have been well documented in repetitive DNA families (see Dover 1982 for review). Origin of Laboratory Mice The 15 inbred strains examined so far possess the domesticus type NTS: r 1 and r2. This is consistent with the comparative analysis of chromosomal C-banding patterns (Moriwaki et al. 1982, 1985) and endonuclease-cleavage patterns of mtDNA (Yonekawa et al. 1980, 1982; Ferris et al. 1982, 1983b), which suggested that the whole mitochondrial genomes and most of the nuclear genes of inbred mice were derived from western Europe (M. m. domesticus). Some nuclear genes appear, however, to have originated in Asiatic mice (M. m. molossinus and others). Further, it is interesting to speculate on the origin of restriction fragments found in several of the inbred strains as minor components. For example, PL/J had 5.5-kb DNA, and AU/SsJ and RFM/ MS contained 7.0-kb DNA on EcoRI digestion. This suggests that brevirostris contributed to the establishment of the PL/J strain. However, it is not clear at present whether the 7.0-kb fragment found in AU/&J and RFM/Ms should be included in the category of r8 or in one of the other NTS haplotypes not used in the present study. Double digestion of 7.0-kb DNA with EcoRI and BamHI suggested the latter possibility (data not shown). Acknowledgments

We are grateful to Dr. T. Gojobori were kindly supplied by Dr. J. P. Hjorth was supported in part by Grants-in-Aid Education, Science and Culture, Japan. National Institute of Genetics, Japan.

for helpful discussions. The mice, MUS-NJL, (University of Aarhus, Denmark). This study for Scientific Research from the Ministry of This paper is contribution no. 1640 from the

136 Suzuki, Miyashita, Moriwaki, et al. LITERATURE CITED ARNHEIM, N. 1983. Concerted evolution of multigene families. Pp. 38-61 in M. NEI and R. K. KOEHN, eds. Evolution of genes and proteins. Sinauer, Sunderland, Mass. ATWOOD, K. C., S. GLVECKSOHN-WAELSH,M. T. Yu, and A. S. HENDERSON.1976. Does the T-locus in the mouse include ribosomal DNA? Cytogenet. Cell Genet. 17:9- 17. BONHOMME,F., J. BRITTON-DAVIDIAN,L. THALER, and C. TRIANTAPYLLIDIS.1978. Sur l’existence en Europe de quatre groups de souris (genre Mus L.) du rang esp&e et semi-esp+ce, demontree par la genetique biochemique. C. R. Acad. Sci. (Paris), ser. D, 287:631-633. BONHOMME,F., J. CATALAN,J. BRI-ITON-DAVIDIAN,V. M. CHAPMAN,K. MORIWAIU,E. NEVO, and L. THALER. 1984. Biochemical diversity and evolution in the genus A4us. Biochem. Genet. 22:275-303. BOURSOT,P., F. BONHOMME,J. BRITTON-DAVIDIAN,J. CATALAN,H. YONEKAWA,P. ORSINI, S. GUERASSIMOV,and L. THALER. 1984. Introgression differentielle des genomes nucleaires et mitochondriaux chez deux semiespeces europeennes de souris. C. R. Acad. Sci. (Paris) 299:365-370. CORRY, S., and J. M. ADAMS. 1977. A very large repeating unit of mouse DNA containing the 18S, 28s and 5.8s rRNA genes. Cell 11:795-805. DOVER, G. 1982. Molecular drive: a cohesive mode of species evolution. Nature 299: 11l-l 17. FERRIS, S. D., R. D. SAGE, C.-M. HUANG, J. T. NIELSEN,U. RITTE, and A. C. WILSON. 1983~. Flow of mitochondrial DNA across a species boundary. Proc. Natl. Acad. Sci. USA 80:22902294. FERRIS, S. D., R. D. SAGE, E. M. PRAGER, U. RITTE, and A. C. WILSON. 1983b. Mitochondrial DNA evolution in mice. Genetics 105:68 l-72 1. FERRIS, S. D., R. D. SAGE, and A. C. WILSON. 1982. Evidence from mtDNA sequences that common laboratory strains of inbred mice are descended from a single female. Nature 295: 163-165. HUCHINSON,C. A., J. E. NEWBALD,S. S. POTTER,and M. M. EDGELL.1974. Maternal inheritance of mammalian mitochondrial DNA. Nature 251:536-538. KOMINAMI,R., Y. MISHIMA, Y. URANO, M. SASAKI,and M. MURAMATSU. 1982. Cloning and determination of the transcription termination site of ribosomal RNA gene of the mouse. Nucleic Acids Res. 10: 1963- 1979. KOMINAMI,R., Y. URANO, Y. MISHIMA,and M. MURAMATSU.1981. Organization of ribosomal RNA gene repeats of the mouse. Nucleic Acids Res. 9:3219-3233. KUEHN, M., and N. ARNHEIM. 1983. Nucleotide sequence of the genetically labile repeated elements 5’to the origin of mouse rRNA transcription. Nucleic Acids Res. 11:2 1 l-224. MANIATIS,T., E. F. FRITSCH, and J. SAMBROOK.1982. Molecular cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. MIYASHITA,N., K. MORIWAIU, M. MINEZAWA,Z. Yu, D. Lu, S. MIGITA, H. YONEKAWA,and F. BONHOMME.1985. Allelic constitution of hemoglobin beta chain in wild populations of the house mouse, A4u.s musculus. Biochem. Genet. 23:975-986. MORIWAKI, K., N. MIYASHITA, and H. YONEKAWA. 1985. Genetic survey of the origin of laboratory mice and its implication in genetic monitoring. Pp. 237-247 in hW-IBALD, DITCHFIELD,and ROWSELL,eds. The contribution of laboratory animal science to the welfare of man and animals. Fischer, Stuttgart. MORIWAKI, K., T. SHIROISHI, H. YONEKAWA,N. MIYASHITA, and T. SAGAI. 1982. Genetic status of Japanese wild mice and immunological characters of their H2 antigens. Pp. 157175 in T. MURAMATSU,G. GACHELIN,A. A. MOSCONA,and Y. IKAWA,eds. Teratocarcinoma and embryonic cell interactions. Japan Scientific Societies, Tokyo, and Academic Press, New York. SAGE, R. S. 198 1. Wild mice. Pp. 39-90. in H. L. FOSTER, J. D. SMALL,and J. G. Fox, eds. The mouse in biomedical research. Vol 1. Academic Press, New York.


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SCHWARTZ,E., and H. K. SCHWARZ.1943. The wild and commensal stocks of the house mouse, MUS musculus Linnaeus. J. Mammal. 24:59-72. SOUTHERN,E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-5 17. THALER, L., F. BONHOMME,and J. BRITTON-DAVIDIAN. 1981. Processes of speciation and semi-speciation in the house mouse. Symp. Zool. Sot. (Lond.) 47:27. WILSON, G. N., M. KNOLLER, L. L. SZURA, and R. D. SCHMICKEL. 1984. Individual and evolutionary variation of primate ribosomal DNA transcription initiation regions. Mol. Biol. Evol. 1:22 l-237. YONEKAWA, H., K. MORIWAIU, 0. GOTOH, J. I. HAYASHI, J. WATANABE,N. MIYASHITA, M. L. PETRAS, and Y. TAGASHIRA. 198 1. Evolutionary relationships among five subspecies of A4u.s musculus based on restriction enzyme cleavage patterns of mitochondrial DNA. Genetics 98:80 l-8 16. YONEKAWA,H., K. MORIWAIU, 0. GOTOH, N. MIYASHITA,S. MIGITA, F. BONHOMME,J. P. HJORTH, M. L. PETRAS,and Y. TAGASHIRA.1982. Origins of laboratory mice deduced from restriction patterns of mitochondrial DNA. Differentiation 22:222-226. YONEKAWA,H., K. MORIWAKI, 0. GOTOH, J. WATANABE,J. I. HAYASHI, N. MIYASHITA, M. L. PETRAS, and Y. TAGASHIRA. 1980. Relationship between laboratory mice and the subspecies Mus musculus domesticus based on restriction endonuclease cleavage patterns of mitochondrial DNA. Jpn. J. Genet. 55:289-296. MASATOSHI

Received

NEI, reviewing

August

editor

6, 1985; revision

received

September

30, 1985.