Proc. Natl. Acad. Sci. USAVol. 77, No. 6, pp. 3595-3599, June 1980Genetics

Comparative gene mapping: Order of loci on the X chromosome isdifferent in mice and humans*

[somatic cell hybridization/Searle's T(X;16)16H translocation/a-galactosidase/hypoxanthine phosphoribosyltransferase]

UTA FRANCKE AND R. THOMAS TAGGARTDepartment of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510

Communicated by Victor A. McKusick, March 24,1980

ABSTRACT For comparative studies we have used the so-matic cell hybridization approach to regionally map genes onthe mouse X chromosome. Fibroblasts from a mouse with thebalanced reciprocal translocation T(XD;16B5)16H were fusedwith a Chinese hamster cell line (V79/380-6) deficient in activityof the enzyme hypoxanthine phosphoribosyltransferase (HPBRTTInterspecific ceI hybrids were initially selected for retentionof the mouse translocation chromosome carrying the Hprt gene.Subsequently, hybrid clones were counterselected to forcesegregation of this chromosome. Selected and counterselectedhybrid clones were analyzed for their chromosome content bytsin/Giemsa banding and for expression of the mouse formsof the X-linked enzymes HPRT and a-galactosidase (GALA) byisoelectric focusing. The results indicate that the breakpoint onthe mouse X chromosome (in band XD) has separated thegenesfor HPRT (Hprt) and for GALA (Ags). fHprt is proximal to thebreakpoint in region Xcen-XD and Ags is distal in regionXD-Xter. The gene order in the mouse (centromere-Hprt-Ags)is therefore inverted when compared to the order of the ho-mologous loci on the long arm of the human X (centromere-GALA-HPRT.Studies in comparative mammalian cytogenetics have dem-onstrated great variability in karyotypes. Diploid chromosomenumbers range from 6 to 84, and for distantly related mam-malian species there are striking differences in chromosomemorphology and banding patterns (1). An exception is providedby the X chromosome, which has been largely conserved (2, 3).The establishment of a dosage compensation mechanism in amammalian ancestor that assures hemizygous expression of Xchromosomal genes in somatic cells may have placed restrictionson further rearrangements between the X chromosome andautosomes during evolution (4). The X chromosome, carryinga large number of gene loci not involved in sex determination,was thus conserved in its entirety (Ohno's hypothesis). Evidencein support of the hypothesis has come from the cytologicalobservation that in mammalian species the X chromosomeuniformly makes up 5-6% of the haploid set (3). Strongestsupport has been provided by comparative gene mappingstudies. The homologous enzyme loci discussed in this reporthave been assigned to the X chromosome in as many as 15mammalian species (5). No gene that is X-linked in one specieshas been found to be autosomal in another. For the comparativestudy of karyotype evolution in distantly related species, suchas humans and mice, the X syntenic group offers a distinctiveadvantage in that homologous genes have been conserved ona single chromosome. Morphologic and genetic evolutionarychanges should be limited to intrachromosomal rearrangements.It is of interest to know to what degree the gene order and rel-ative distances between loci have been conserved. One wouldexpect that X-chromosomal loci are not arranged randomly butthat a certain gene order might be advantageous, or even es-sential. On the other hand, the cytologic observation that

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3595

mammalian X chromosomes are by no means identical incentromere position and banding patterns suggests that someintrachromosomal rearrangements, such as inversions and shifts,have occurred. Comparing the X chromosome maps of dif-ferent mammals may allow one to trace these evolutionarysteps.

This report concentrates on enzyme loci that are expressedin cultured cells, for which homology in mice and humans hasbeen established, and whose assignments to the mouse Xchromosome have been made or confirmed by somatic cellgenetic studies: a-D-galactosidase (GALA, gene GALA or Ags)(6, 7) hypoxanthine phosphoribosyltransferase (HPRT, geneHPRT or Hprt) (7-9), and phosphoglycerate kinase 1 (PGK-1,gene Pgk-1) (8, 10).

Limited information is available on regional mapping andorder of X-linked genes. In Mus musculus the structural locifor PGK-1 (Pgk-1) and GALA (Ags) have been mapped bygenetic studies using an electrophoretic variant of PGK-1 (11)and a thermolabile variant of GALA (12). The resulting geneorder is cen (centromere)-Pgk-l-Ags. The genes for HPRT(Hprt) and glucose-6-phosphate dehydrogenase (G6PD, geneC6pd) have been assigned to the mouse X chromosome by genedosage studies in embryos (13, 14), and the assignments havebeen confirmed by somatic cell genetic studies, including so-matic cell hybridization (7-9). Because no variants for HPRTand G6PD have as yet been found in M. musculus, the mappositions of these loci are unknown. Gene assignments to cy-tologically defined regions of the mouse X chromosome havenot yet been reported.

In humans, however, regional localizations of the four en-zyme loci on the long arm of the X chromosome have been es-tablished by somatic cell genetic experiments. The order iscen-PGK-GALA-HPRT-G6PD. HPRT and G6PD are bothlocated in the most distal band, Xq28 (15). The successful ap-proach for mapping of the human X has been the introductionof X/autosome translocations into somatic cell hybrids segre-gating human chromosomes.

Following that same strategy for regional mapping of locion the mouse X chromosome, we have used the reciprocalX/autosome translocation T(X;16)16H known as Searle'stranslocation. Previous meiotic studies had demonstrated thatthis translocation involves a reciprocal exchange between thedistal half of a small autosome and the distal third of the Xchromosome (16). Genetic studies placed the T16H breakpointbetween the genes Bn (bent tail) and Ta (tabby) on the Xchromosome (17). Quinacrine mustard banding studies iden-

Abbreviations: HPRT, hypoxanthine phosphoribosyltransferase (EC2.4.2.8); GALA, a-D-galactosidase (EC 3.2.1.22); PGK, phosphoglyc-erate kinase (EC 2.7.2.3); SOD, superoxide dismutase (EC 1.15.1.1);G6PD glucose-6-phosphate dehydrogenase (EC 1.1.1.49); HAT, hy-poxanthine/aminopterin/thymidine; 8-AG, 8-azaguanine; 8-AGR,8-AG-resistant; Ags, gene for a-galactosidase in the mouse.* This work was presented at the Vth International Workshop onHuman Gene Mapping, Edinburgh, Scotland, July 1979.

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3596 Genetics: Francke and Taggart

tified chromosome 16 as the autosome involved (18). We haverestudied the translocation with trypsin/Giemsa banding andhave defined breakpoints in bands 16B5 and XD (19).

Somatic cell hybrids between an established Chinese hamstercell line and diploid mouse cells segregate mouse chromosomesand are useful for gene assignments in the mouse (6, 7). We havehybridized fibroblasts containing the T16H translocation to anHPRT-deficient Chinese hamster cell line. Growth of hybridcells in the appropriate selective media will force retention orsegregation of the part of the mouse X chromosome that carriesthe gene for HPRT. We have previously reported our resultsbased on cell selection that have placed Hprt on X16 (containingregion Xcen-XD) and the assignment of the gene for cyto-plasmic superoxide dismutase (SOD, gene Sod-i) to the distalpart of chromosome 16 (region 16B5-16ter contained inchromosome X16) (19). We have now studied the expression ofmouse GALA and HPRT activity in hybrid clones that containdefined regions of the mouse X and that are therefore infor-mative for regional mapping. GALA expression did not seg-regate concordantly with Hprt, indicating that the translocationbreakpoint in band XD has separated these two loci. The re-sulting gene order cen-Hprt-Ags differs from the order inhumans and suggests the occurrence of intrachromosomal re-arrangements, possibly a pericentric inversion or a shift of thecentromere. However, when all currently available data aretaken together, the gene order on the mouse X is cen-Hprt-Pgk-1-Ags, whereas on the human X long arm it is cen-PGK-GALA-HPRT. Comparison of the gene order in the twospecies suggests that the evolutionary rearrangements have beenmore complex than a single inversion.

MATERIALS AND METHODSCell Hybrids. Somatic cell hybrids were produced by Sendai

virus-mediated fusion of primary fibroblasts from an adult malemouse with Searle's T(X;16)16H translocation to an establishedChinese hamster cell line (V79, clone 380 deficient in HPRTactivity) (19). Cell hybrid clones were selected and maintainedin medium containing hypoxanthine, aminopterin, thymidine,and glycine (HAT) (20). In the T16H reciprocal translocation

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the distal third of the X chromosome has been exchanged withthe distal half of chromosome 16; therefore, continuous growthof hybrids in HAT medium will select for retention of eitherX16 or 16X, depending on the location of the mouse HPRT gene.In contrast, hybrids able to grow in the presence of the guanineanalog 8-azaguanine (8-AG) must have lost HPRT activity,presumably due to segregation of the HPRT-bearing translo-cation chromosome. Five primary independent hybrid clonescontaining both translocation chromosomes while in HATmedium were plated at low density in nonselective medium.Subsequently, 15 secondary clones were isolated in mediumcontaining 20 ,uM 8-AG. They are referred to as 8-AG-resistant(8-AGR) clones (19).

Electrophoresis. The procedure for obtaining cell extractshas been described (19). GALA, HPRT, and SOD isozymeswere separated by isoelectric focusing in horizontal sheetpolyacrylamide gels. The 1.5 mm X 18 cm X 18 cm gels con-tained 4.8% acrylamide, 0.2% N,N'-methylenebisacrylamide,20% (vol/vol) glycerol, and a specific ampholyte mixture foreach enzyme. The GALA gel contained 0.8% pH 4-6.5 and1.2% pH 2.5-5.0 ampholytes (Pharmacia). A pH 4-6.5 am-pholyte solution (2.0%) was used for the cathode and 1.0 MH3P04 was used for the anode. The HPRT gel contained 2.0%pH 5-8 ampholytes. The SOD gel contained 0.8% pH 3-10 and1.2% pH 5-8 ampholytes. The cathode solution for the HPRTand SOD focusing gels was 1.0 M NaOH. The anode solutionwas 1.0M HSP04 for SOD and 0.02M glutamic acid for HRPT.The method of electrofocusing, including the conditions ofprefocusing and sample application and the detection of SODisozymes, was as described (19).GALA activity was detected after an initial equilibration of

the gel in 0.1 M citric acid/phosphate buffer (pH 4.0, 10 min)followed by incubation at 37°C with filter paper saturated in0.1 M citric acid/phosphate containing 4-methylumbellif-eryl-a-D-galactoside (Sigma) at 1 mg/ml. Areas of GALA ac-tivity were fluorescent under long-wave UV illumination andwere intensified by exposure to alkaline pH (21).HRPT activity was detected by equilibratioh of the gel in 0.1

M Tris.HCl (pH 7.4) followed by incubation at 370C with filter

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paper saturated in a solution containing 0.1 M Tris-HCl, 10 mMMgCl2, 0.25 mM phosphoribosylpyrophosphate, and [8-14C]-hypoxanthine (New England Nuclear) at 1 uCi/ml (1 Ci = 3.7X 1010 becquerels). The filter paper was removed after 45 minand the gel was rinsed briefly in 0.1 M Tris.HC1 before exposureto 0.1 M LaCl3 for 1 hr, which precipitated the [14C]inosinemonophosphate reaction product (21). The gel was rinsed twicein distilled H20 and dried onto filter paper with a gel dryer(Bio-Rad). The areas containing HPRT activity were visualizedby exposure of the gel to Kodak NS-2T X-ray film for 24 hr.M. musculus and Chinese hamster (cell line 380) isozymes

of PGK and G6PD were not separable by isoelectric focusing.Chromosome analysis. The chromosome constitution of the

hybrid clones used for this study was determined on differentoccasions, at the same passage or a closely related passage to theones at which enzyme studies were carried out. Cells wereharvested for chromosome analysis as described (7). Thetrypsin/Giemsa banding procedure was the same as reportedfor human chromosomes (22). The mouse centromeric heter-ochromatin stains vary darkly with this technique, allowing easyidentification of mouse chromosomes and eliminating the needfor routine staining with Hoechst 33258.The frequency of each mouse chromosome was determined

by karyotyping 10-31 metaphase spreads from each clone, andis expressed as the average number of the respective chromo-some per metaphase cell.

RESULTSAll hybrid clones selected in HAT medium had retained bothT16H translocation chromosomes X16 and 16X and, therefore,were not informative for regional mapping of X-linked genes(Fig. 1). In contrast, all the back-selected 8-AGR subclones hadlost X16, while the reciprocal translocation product 16X wasretained in several 8-AGR subclones at frequencies ranging from0.13 to 1.1 (Fig. 2).GALA activity occurs in electropherograms of cell extracts

as multiple bands, presumably due to posttranslational modi-fications (21). In our isoelectric focusing system, Chinesehamster (cell line 380) activity migrated more anodally (Fig.

3 Upper, lane 1) than mouse (BALB/3T3) activity which cov-ered a wide range (lane 2). Mouse (T16H)-Chinese hamster(380) hybrid clones expressing mouse GALA (lane 3) couldeasily be distinguished from those lacking mouse GALA activity(lane 4).Mouse and Chinese hamster (V79) HPRT, were detected on

autoradiograms after isoelectric focusing as partially overlap-ping but distinctly different sets of bands (Fig. 3 Lower). Theparental Chinese hamster line 380 contained no HPRT activity.All hybrid clones grown in HAT medium produced the mousepattern of HPRT activity, whereas all 8-AGR subclones lackedHPRT activity.

Table 1 shows the relative frequencies of X16 and 16X com-pared to the expression of mouse HPRT, GALA, and SOD-1activities [the latter being a marker for X16 (19)] in a series ofhybrid clones. While the primary HAT clones containing bothtranslocation products were positive for the three mouse iso-zymes, in all 8-AGR subclones the X16 chromosome had beenlost concordantly with HPRT activity (and with mouse SOD-Iexpression in clones without the normal chromosome 16).However, mouse GALA activity segregated concordantly withthe other translocation product 16X. A single exception wasclone 4A1-2a-AGR, which was GALA positive but did notcontain 16X. A new rearranged chromosome was discoveredof size and banding pattern consistent with its derivation from16X. This rearrangement was not present in the GALA-negativesister clone 4A1-2c-AGR. Clone 22A2 HAT is an example forthe limits of detection of activity; weak staining of the mouseGALA bands is correlated with a low frequency (0.13) of the16X chromosome. These results allow the conclusion that thebreak in the mouse X chromosome leading to the T16H trans-location has separated the genes for HPRT (Hprt) and GALA(Ags): Hprt is proximal and Ags is distal.

DISCUSSIONUsing fibroblasts from a murine reciprocal X/autosometranslocation (T16H) carrier for somatic cell hybridization, wehave obtained hybrid clones that were informative for regionalassignments of three gene loci in the mouse: Sod-i to 16B5-

Genetics: Francke and Taggart

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3598 Genetics: Francke and Taggart

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tivity. Lane 1, Chinese hamster cell line 380-6, HPRT deficient; 2,mouse cell line BALB/3T3; 3, Chinese hamster (380)-mouse (T16H)somatic cell hybrid clone expressing both types of activity (M, mouse;CH, Chinese hamster); 4, 380-T16H hybrid clone with only Chinesehamster activity. (Lower) Patterns of HPRT activity obtained byisoelectric focusing and autoradiography. Lane 1, hybrid clone 18AHAT; 2, 380-5R, HPRT-positive revertant ofV79/380 mutant Chinesehamster cell line; 3, primary diploid Chinese hamster fibroblasts; 4,mouse control, cell line L929. The hybrid in lane 1 has the mouse

pattern of HPRT activity, lacking the most basic (top) Chinesehamster band.

16ter, Hprt to Xcen-XD, and Ags to XD-Xter. The significanceof localizing Sod-i as a possible marker for the genes causingDown syndrome in humans has been discussed elsewhere (19).The present report focuses on the order of loci on the mouse

X chromosome. The location of Hprt on X16 had been inferredfrom the observation that X16 was not present in 8-AGR sub-

clones. We have demonstrated here that 8-AGR clones thatretained the other translocation chromosome 16X were indeedlacking HPRT activity. Such clones, however, did express

mouse GALA activity. The resulting localization of Ags to re-

gion XD-Xter (23) is consistent with our earlier observation ofa Chinese hamster-mouse hybrid clone that expressed mouseHPRT but not GALA and that contained an abnormal Xchromosome with deletion of the distal bands XE and XF (7).On the linkage map of the mouse X chromosome, the pre-sumably structural gene for GALA (AgS) maps distal to theT16H breakpoint, as determined by pedigree studies using a

mouse strain with a thermolabile GALA variant (12). Geneticand cytological maps are in agreement if one assumes that the

Table 1. Frequency of mouse chromosomes 16, X16, and 16X andexpression of mouse enzymes SOD-1, HPRT, and GALA in sixprimary mouse (T16H)-Chinese hamster (380) hybrids and in

eight 8-AGR subclonesHybrid Mouse chromosomes and enzyme expressionclone SOD-1 16 X16 16X HPRT GALA

3-2D HAT + 0.6 1.2 1.1 + +3-2D-lb 8-AGR - 0 0 0.9 - +3-2D-lc 8-AGR - 0 0 0.8 - +4A1 HAT + 1.0 0.9 1.0 + +4A1-2a 8-AGR + 1.3 0 0* - +4A1-2c 8-AGR + 1.6 0 0 - -10C HAT + 0 1.0 1.1 + +10C-ic 8-AGR - 0 0 0.7 - +13A HAT + 0.4 0.9 0.6 + +13A-la 8-AGR + 1.1 0 0.8 - +13A-3a 8-AGR + 0.8 0 018A HAT + 0 1.0 1.0 + +18A-1a 8-AGR - 0 0 022A2 HAT + 0.4 0.8 0.13 + (+)

The frequency of mouse chromosomes is expressed as averagenumber of copies per cell. +, Mouse enzyme expressed; -, absent; (+),weakly positive.* This 8-AGR subclone contained a new rearranged chromosomeprobably derived from 16X.

thermolabile variant and the bands of enzyme activity pro-duced by somatic cell extracts are products of the same gene.The structural gene for PGK-1 (Pgk-1) has also been mapped

distal to the T16H breakpoint, but proximal to Ags, by geneticstudies of a mouse strain with an electrophoretic PGK variant(11). The linkage map position of G6pd is not known becauseno variants have been detected as yet. Regional assignments ofthese two loci by somatic cell genetics was not successful becausewe were unable to separate the mouse and Chinese hamsterphenotypes for G6PD and PGK.A major unsolved problem in gene mapping concerns the

relationship between cytologic and genetic maps. In the mouse,the linkage map is extensive and detailed, and translocationbreakpoints have been mapped by genetic studies. Correlationbetween linkage map position and cytologic position of trans-location breakpoints has allowed the assignment of gene locito specific chromosome bands (23). On the basis of such acomparison, the genetic and cytologic maps for mouse chro-mosome 2 have been shown to be colinear (24). The map dis-tances between loci, however, are not expected to be propor-tional because chiasmata are nonrandomly distributed, theirlocation being determined by size of the chromosome and in-terference by other chiasmata (25). The genetic mapping oftranslocation breakpoints is complicated by changes in crossoverfrequency in the vicinity of the breakpoints. Therefore, regionalmapping data derived independently from somatic cell hybridssegregating translocation chromosomes add valuable infor-mation of the correlation of both kinds of gene maps.

In Fig. 4 the regional assignments of genes on the long armof the human X chromosome are shown in comparison to theregional assignments on the mouse X, as reported here, and tothe mouse linkage map (26). Whereas the gene order on thehuman X is cen-PGK-GALA-HPRT, in the mouse it is cen-Hprt-Pgk-1-Ags, assuming colinearity between genetic andcytological maps. These differences are not explained by asingle inversion or shift of the centromere. A simple shift of theband containing Hprt seems unlikely in view of the differencesin positions of the centromere and in banding patterns. Severalsteps of intrachromosomal rearrangements must have occurredduring evolution separating mice and humans. One hypothesisinvolves at least two events: inversion of a large part of the Xthat moved the centromere and allowed the banding pattern

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FIG. 4. (A) Diagram of G-banding pattern of the human X chromosome (23) with regional localizations of enzyme loci (15). (B) Ideogramof G-banding pattern of the mouse X chromosome (20). Arrow indicates breakpoint in T(XD;16B5)16H translocation (19). Brackets indicateregional assignments for Hprt and GALA (Ags) reported here. (C) Partial linkage map of the mouse X chromosome (26). Bracket on the rightdenotes map position of the T16H breakpoint. The centromeres are at the top in B and C.

to remain similar and a small shift or inversion that reversedthe order of GALA and PGK.An alternate hypothesis would be that the T(X;16)16H

translocation was not a simple reciprocal exchange of terminalregions but rather involved interstitial segments. If Hprt were

located in the terminal band XF4 (in analogy to the humanHPRT in Xq28), it might have been incorporated into X'6 incase of an interstitial translocation. The gene order, in this case,would have been conserved. However, meiotic studies havestrongly suggested the existence of a simple reciprocal exchange(16), in agreement with impressions from mitotic chromosomebanding studies (18, 19). We, therefore, believe that our resultsprovide evidence that the gene order of homologous enzyme

loci, not closely linked on the X chromosome, is different inmice and humans. While evolutionary pressure has conservedthe X syntenic group in mammals, restructuring within the Xchromosome that led to changes in centromere positions, inbanding patterns, and in gene order has occurred. More detailedstudies are needed to determine whether there are limits to thispermissiveness and whether there are favored patterns of in-trachromosomal rearrangements in the evolution of mamma-lian X chromosomes.

Dr. Eva Eicher suggested this experiment in 1972. Dr. J. A. Schneiderand Ms. 0. Pellett gave expert advice on cell fusion and providedlaboratory facilities. M. G. West, E. Rockwell, R. Habermann, A.Davidoff, and P. Tetri provided technical assistance. The work was

supported by Research Grants GM-21110 and GM-26105 from theNational Institutes of Health.

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2. Ohno, S. (1967) Sex Chromosomes and Sex-Linked Genes(Springer, Heidelberg, West Germany).

3. Ohno, S. (1969) Annu. Rev. Genet. 3, 495-524.4. Ohno, S. (1973) Nature (London) 244, 259-262.

5. Pearson, P. L. & Roderick, T. H. (1979) Cytogenet. Cell Genet.25,82-95.

6. Kozak, C., Nichols, E. & Ruddle, F. H. (1975) Somatic CellGenet. 4, 371-382.

7. Francke, U., Lalley, P. A., Moss, W., Ivy, J. & Minna, J. D. (1977)Cytogenet. Cell Genet. 19, 57-84.

8. Chapman, V. M. & Shows, T. B. (1976) Nature (London) 259,665-667.

9. Hashmi, S. & Miller, 0. J. (1976) Cytogenet. Cell Genet. 17,35-41.

10. Kozak, L. P., McLean, G. K. & Eicher, E. M. (1974) Biochem.Genet. 11, 41-47.

11. Nielsen, J. T. & Chapman, V. M. (1977) Genetics 87, 319-325.

12. Lusis, A. M. & West, J. D. (1976) Biochem. Genet. 14, 849-855.

13. Epstein, C. J. (1969) Science 163, 1078-1079.14. Epstein, C. J. (1972) Science 175, 1467-1468.15. Miller, 0. J., Sanger, R. & Siniscalco, M. (1978) Cytogenet. Cell

Genet. 22, 124-128.16. Lyon, M. F., Searle, A. G., C. E. & Ohno, S. (1964) Cytogenetics

3,306-323.17. Lyon, M. F. (1966) Genet. Res. 7, 130-133.18. Eicher, E. M., Nesbitt, M. N. & Francke, U. (1972) Genetics 71,

643-648.19. Francke, U. & Taggart, R. T. (1979) Proc. Natl. Acad. Sci. USA

76,5230-5233.20. Littlefield, J. W. (1964) Science 45, 709-710.21. Harris, H. & Hopkinson, D. A. (1976) Handbook of Enzyme

Electrophoresis in Human Genetics (Elsevier, New York).22. Francke, U. & Oliver, N. (1978) Hum. Genet. 45, 137-165.23. Nesbitt, M. N. & Francke, U. (1973) Chromosoma 41, 145-

158.24. Searle, A. G., Beechey, C. V., Eicher, E. M., Nesbitt, M. N. &

Washburn, L. L. (1979) Cytogenet. Cell Genet. 23,255-263.25. Lyon, M. F. (1976) Genet. Res. 28,291-299.26. Davisson, M. T. & Roderick, T. H. (1979) Mouse Newsl. 61,

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