Molecular Markers for the agouti Coat Color ... - Genetics · Molecular Markers for the agouti Coat Color Locus of the Mouse Michael Lovett, Zai-yu Cheng,' Estrella M. Lamela, Tohru

Copyright 0 1987 by the Genetics Society of America

Molecular Markers for the agouti Coat Color Locus of the Mouse

Michael Lovett, Zai-yu Cheng,' Estrella M. Lamela, Tohru Yokoi* and Charles J. Epstein Department of Pediatrics and of Biochemistry and Biophysics, University of Calqornia, San Francisco, Calyornh 941 4 3

Manuscript received September 3, 1986 Revised copy accepted December 22, 1986

ABSTRACT The agouti (a) coat color locus of the mouse acts within the microenvironment of the hair follicle

to control the relative amount and distribution of yellow and black pigment in the coat hairs. Over 18 different mutations with complex dominance relationships have been described at this locus. The lethal yellow (AY) mutation is the top dominant of this series and is uniquely associated with an endogenous provirus, Emv-15, in three highly inbred strains. However, we report here that it is unlikely that the provirus itself causes the Ay-associated alteration in coat color, since one strain of mice (YBR-Ay/a) lacks the provirus but still retains a yellow coat color. Using single-copy mouse DNA sequences from the regions flanking Emv-I5 we have detected three patterns of restriction fragment length polymorphisms (RFLPs) within this region that can be used as molecular markers for different agouti locus alleles: a wild-type agouti (A) pattern, a pattern which generally cosegregates with the nonagouti (a) mutation, and a pattern which is specific to Emv-15. We have used these RFLPs and a panel of 28 recombinant inbred mouse strains to determine the genetic linkage of these sequences with the agouti locus and have found complete concordance between the two (95% confidence limit of 0.00 to 3.79 centimorgans). We have also physically mapped these sequences by in situ hybridization to band HI of chromosome 2, thus directly confirming previous assignments of the location of the agouti locus.

HE agouti (a) coat color locus of the mouse is T located on chromosome 2 and affects the relative amount of the two pigments phaeomelanin (yellow pigment) and eumelanin (black pigment) and their distribution within the coat of the mouse (SILVERS 1979). Alleles at this locus appear to affect the microenvironment of the hair follicle, which in turn alters the behavior of hair bulb melanocytes (SILVERS 1958). In wild-type agouti (A/A) mice, a switch from the deposition of eumelanin to phaeomelanin and back to eumelanin occurs within individual melanocytes. This results in a charPcteristic subapical band of yellow pigmentation on an otherwise black or brown hair. Over 18 different mutations have been described at the agouti locus. At opposite ends of this series are the bottom recessive allele, extreme nonagouti (ac/ac), which produces only black pigment, and the top dominant allele, lethal yellow (AY/+), which produces only yellow pigment. Some alleles also produce alterations in the pattern of pigment deposition. For example, animals homozygous for the black and tan allele (a'/.') exhibit a black dorsum and a yellow- colored ventrum.

Recombination between some agouti locus alleles has been reported (RUSSELL, MCDANIEL and WOODIEL 1963), and some controversy exists as to whether all

Present address: Department of Medical Genetics, Institute of Basic

' Present address: Department of Pediatrics, Kanazawa University School Medical Sciences, Chinese Academy of Medical Science, Ekijing, China.

of Medicine, 13-1, Takaramachi, Kanazawa, Ishikawa, Japan.

Genetics 115: 747-754 (April, 1987)

of the mutations at this locus are truly allelic or pseudoallelic [see SILVERS (1979) for a discussion of this point]. One mutation which affects agouti locus function, the agouti suppressor (A') mutation (PHIL- LIPS 196 l) , is particularly interesting since it appears to exert its action over a substantial genetic distance. This position effect mutation, which is a radiation- induced chromosomal inversion [Zn(2)2H] exhibits 0.6% recombination with the agouti locus and sup- presses the effects of the agouti allele to which it is linked (PHILLIPS 1966; EVANS and PHILLIPS 1978). It is therefore possible that the agouti locus may be both large and complex.

Several mutations at the agouti locus can result in phenotypic abnormalities above and beyond alterations in pigment deposition. Four alleles are prenatal homozygous lethals (AY, a', ax and (EATON and GREEN 1963; LYON, FISHER and GLENISTER 1985; PAPAIOANNOU and MARDON 1983). While AY/a' mice die during embryogenesis (LYON, FISHER and GLEN- ISTER 1985), these mutations are clearly not all mem- bers of the same lethal complementation group, since

and AY/a" mice are fully viable. In addition, the AY and A"J alleles, the former lethal when homozygous, the latter not, both produce a yellow coat color, maturity-onset obesity, an increased tumor in- cidence and various other metabolic abnormalities, the biochemical bases of which are unknown (WOLFF 1970; WOLFF and PITOT 1973; WOLFF, ROBERTS and GALBRAITH 1986).

748 M. Lovett et al.

T h e AY allele is unique among all the agouti locus alleles in being associated with an endogenous ecotropic provirus (Emu-15) in three highly inbred strains (COPELAND, JENKINS and LEE 1983). This observation suggested that isolation of the Emu-I5 integration site might allow entry to be gained to regions close to the agouti locus. Furthermore, the ability of retroviruses to act as insertional mutagens (HAYWARD, NEEL and ASTRIN 198 1 ; VARMUS, QUINTRELL and ORTIZ 198 1 ; COPELAND, HUTCHISON and JENKINS 1983; SCHNIEKE, HARBERS andJAENIsCH 1983) suggested that the Emu- 15 provirus might have caused one or more of the altered phenotypic features which are associated with the AY mutation.

We have recently isolated molecular clones of the Emu-I5 proviral integration site and have detected transcriptionally active mouse DNA sequences downstream from the provirus (LOVETT and EPSTEIN, 1987). In mice heterozygous for the AY mutation (either C57BL/6J-AY/a or 1 29/Sv-AY/Aw), transcripts from this region contain both host and viral sequences which are probably initiated from within the provirus. These results suggest that Emu-I5 has altered a mouse gene by promoter insertion (HAYWARD, NEEL and ASTRIN 1981). However, it is not clear what degree of physical and genetic linkage underlies the observed association of the Emu-I5 provirus with the AY allele of the agouti locus. Therefore, the possibility exists that the mutated locus we have identified is merely coincidentally linked to AY and does not give rise to any of the AY-associated phenotypic abnormalities. To determine more accurately the linkage between our DNA markers and the agouti locus, we have genetically mapped these sequences using agouti locus allele- specific restriction fragment length polymorphisms (RFLPs) and have physically mapped them by in situ hybridization to G-banded mouse chromosomes. We report here that the sequences flanking the provirus are indeed closely linked to the agouti locus. However, it is unlikely that the Emu-I5 provirus itself causes the AY-associated alteration in coat color, since a strain of AY/a mice that has lost Emu-I5 still retains a yellow coat color.

MATERIALS AND METHODS

Mouse strains: All mouse strains were obtained from The Jackson Laboratory, Bar Harbor, Maine, with the exception of the following: mice carrying the a', a'6H and A s mutations were provided by Dr. M. LYON, MRC Radiobiology Unit, Harwell, United Kingdom; mice carrying the ax mutation were provided by Dr. L. B. RUSSELL, Oak Ridge National Laboratory, and the YBR/Ki strain was provided by Dr. M. OHMORI, Kirschbaum Memorial Mouse Colony, Kagawa Medical School, Japan.

Recombinant inbred (RI) strains: DNAs from the four RI strains BXHITy, BXJITy, CXB/By and SWXL/Ty were purchased from The Jackson Laboratory. The progenitor strains of these are C57BL/6J (B) and C3H/HeJ (H) for the

BXH strains, C57BL/6J (B) and SJL/J U) for the BXJ strains, C57BL/6J (B) and BALB/cBy (C) for the CXB strains, and SWR/J (SW) and C57L/J (L) for the SWXL strains. The RFLP linkage estimation was performed by the method of SILVER (1 985).

Molecular probes: The pRI subclone contains a 1.1-kb single copy mouse genomic DNA EcoRI fragment, and the pP0.5 subclone contains a 0.5-kb PstI fragment comprised of single copy mouse genomic DNA plus 40 bp (as deduced by DNA sequencing) of DNA from the 5' proviral long terminal repeat (LTR). Both subclones were 32P-labeled (RIGBY et al. 1977) to specific activities of 210' cpm/pg. The pRI subclone was labeled for in situ hybridization by nick translation to specific activities of 1-5 X lo7 cpm/pg using all four 'H-labeled nucleotide triphosphates.

Gel electrophoresis, Southern transfer, and hybridization: High molecular weight DNA was completely digested with the indicated restriction endonucleases under conditions recommended by the manufacturer. Digested DNAs were then electrophoresed in agarose gels in Tris-acetate buffer (40 mM Tris-HC1, 1.0 mM EDTA, 25 mM sodium acetate, pH 7.6). Transfer of DNA to nitrocellulose paper was performed by the method of SOUTHERN (1 975). Filters were hybridized at 65" for 16 hr with 25 ng/ml of 32P- labeled probe DNAs in a solution containing 3 X SSC (1 X SSC is 0.15 M NaC1, 0.015 M sodium citrate, pH 7.0), 0.1% SDS, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albu- min, and 0.2% Ficoll. The filters were washed in 0.1 X SSC with 0.1% SDS at 65" for 1 hr, air dried, and autoradi- ographed.

In situ hybridization: The preparation of metaphase spreads, in situ hybridization, autoradiography and chromosome banding were conducted as previously described (CHENG et al. 1986). Tail fibroblasts were obtained from mice homozygous for the metacentric Robertsonian translocation chromosome, Rb(2. I 7 ) l lRma which is a fusion of mouse chromosomes 2 and 17. Three criteria were used in determining the number of silver grains observed: (1) the chromosome spread was complete, (2) the grains were located on or touched at least one of the two chromatids, and (3) one or more grains at a single site were counted as a single hybridization event.

RESULTS

Emv-15 preinsertion site: The restriction map of the cloned Emu-I5 proviral integration site, the isolation and characterization of which we have described elsewhere (LOVETT and EPSTEIN, 1987), is shown in Figure 1 (top map), as are the positions of two single copy mouse genomic DNA subclones which were derived from it. T h e sequences within one of these subclones, pRI, are part of a mouse transcription unit which is expressed in the same orientation as the proviral genes. In mice heterozygous for AY, transcripts from this region appear to contain viral and host sequences, indicating that the proviral integration has altered this gene. T h e second subclone, pP0.5, contains 40 bp of DNA from the 5' proviral LTR in addition to 500 bp of single copy mouse genomic DNA. Under the hybridization and washing conditions employed in this study this short stretch of proviral sequences does not detectably cross-hybridize with endogenous LTR DNA sequences or with a

Markers for the Agouti Locus 749

BcK 0 EcoRI 8 HindIII D PstI ' P vu11

BamHI San - SStI

0". '. pP0.5 ' ' '. '

A FIGURE 1 .-Restriction maps of the cloned Emu-I5 proviral integration site and preinsertion site. The top map shows the arrangement of

sites within the cloned proviral integration site. The solid bar represents proviral sequences. Below the map are shown the locations of the two subclones, pP0.5 and pRI, the latter of which is 1.1-kb long. The bottom map shows the arrangement of sites within wild type (A/A) DNA as deduced by Southern blotting using the two subclones. Dashed lines show the alignment between these two maps and the arrowhead shows the position of the proviral integration site.

subclone that contains the entire Emv-I5 LTR (data not shown). The lower map in Figure 1 shows the restriction map of the uninterrupted (preinsertion site) genomic DNA in a wild-type (C3H/HeJ-A/A) mouse as deduced by Southern blotting using the two single copy subclones, pP0.5 and pRI (data not shown), as hybridization probes. With the exception of the provirus itself, these two maps exactly align at all sites which we can detect with our two subclones. However, this analysis has the limitation that not all of the sites flanking Emv-15 can be mapped in wild type DNA using our two subclones. It is therefore possible that some small sequence differences exist within the region adjacent to the 3' end of the provirus.

Comparative mapping at sites distal to the integration site: Many of the described agouti locus alleles have been induced by radiation or chemical mutagen- esis. To determine whether any of these alleles might contain substantial additions, rearrangements or dele- tions within the regions surrounding the proviral integration site, we performed Southern blots on DNAs isolated from mice carrying the ae, a', ax, , and A" mutations and also from mice carrying three reciprocal translocations, T( 1;2)5Ca, T(2;8)26H and T(2;11)30H (CARTER, LYON and PHILLIPS 1955; BATCHELOR, PHILLIPS and SEARLE 1966; CATTANACH 1966), the latter two of which affect agouti locus function. All of these translocations have breakpoints within band H 1 of mouse chromosome 2 (EICHER and WASHBURN 1977; DE BOER and VAN GIJSEN 1974; WASHBURN and EICHER 1977) as does the As inversion (Zn[2]2H) (EVANS and PHILLIPS 1978). Using the restriction enzymes EcoRI and BamHI, which cut outside the regions we have cloned (see Figure lB), and probing with both radiolabeled pRI and pP0.5, we have been unable to detect any differences in mobilities between wild-type (A/A) DNA and those listed

above (data not shown). However, the region we can currently investigate using our two subclones is small (-30 kb), and the boundaries of the pRI-homologous transcription unit may extend outside this region. The comparative analysis of sites which are located even further away from the Emu-I5 integration site will require either a "chromosome walk" or the use of more sophisticated gel electrophoretic techniques (CARLE, FRANK and OLSON 1986).

Restriction fragment length polymorphisms: In parallel with the above analysis, we screened a panel of DNAs from mice carrying various agouti locus alleles to determine whether any allele-specific restriction fragment length polymorphisms existed within this region. This screen revealed two RFLPs, both of which are alterations to the sequences between pP0.5 and pRI, within a region which our preliminary RNA mapping data indicate is noncoding. The positions of these polymorphic sites are shown in Figure 2. The polymorphic MspI site immediately adjacent to the proviral integration site can only be identified using pP0.5 as a probe, whereas the polymorphic HindIII site adjacent to pRI can be identified with both probes. We have found two configurations for these sites. In the first (A) configuration, shown in Figure 2B, the HindIII site is present and the MspI site is absent. In the second (B) configuration, the Hind111 site is absent and the MspI site is present (Figure 2C). Some examples of the HindIII polymorphism can be seen in Figure 3, which shows a Southern blot of DNAs from mice carrying various agouti locus alleles digested with HindIII and probed with radiolabeled pRI. The 2.6- kb fragment results from the presence of the HindIII site, the 5.4-kb fragment results from its absence (cJ: Figures 2 and 3).

Some examples of the MsPI polymorphism can be seen in Figure 4, which shows a Southern blot of MspI digested DNAs probed with radiolabeled pP0.5. The

750 M. Lovett et al. H M

pm.5 PRI

S MHPRMMSP HRP R

-...-... A

Site lntegrat/on 5'

S MHPR MSPHRP R

-H si;s B

S MHPRMMSP RP R mH ala c

FIGURE 2.-Restriction maps of RFLP patterns. (A) A composite restriction map of the sites surrounding the insertion site showing the location of the two probes, pP0.5 and pRI. S , Ssd; M, MspI; H, HindI11; P. PstI; R, EcoRI. This arrangement of sites, i.e. the presence of both polymorphic MspI and HindIII sites, does not occur in any strain we have tested and is shown only for comparative purposes. The solid line below the map shows the direction of transcription of the gene of which pRI is a part ( L o m and E m , 1987). Dotted lines reflect the fact that this gene may extend upstream and/or downstream from this point. (B) The wild- type agouti (A/A) map. The presence of the polymorphic HindIII site is shown by an asterisk. (C) The nmugouri @/a) map. The presence of the polymorphic MspI site is shown by an asterisk.

5.4 kb+

2.6 kb +

FIGURE %-Detection of the HindIII RFLP in various mouse strains. A Southern blot is shown of HindIII digested DNAs which were electrophoresed in a 0.8% agarose gel and hybridized with radiolabeled pRI. At the left side the positions of the 2.6-kb (A type) fragment and 5.4-kb (B type) fragment are shown. The strains used in this experiment are, from left to right, CSH/HeJ-A/A, SWR/J-A/A, BALB/cJ-A/A, AKR/Ja/a, DBA/lJa/a, CBA/J-A/A, C57BL/6Ja/a, 1 29/Sv-AJ/AW, 1 29/Sv-Am/A".

1.2-kb fragment results from the presence of the site shown in Figure 2C, the 1.7-kb fragment from its absence. Figure 4 also shows a third MspI RFLP of

1.7 kb+

1.2 kb+

FIGURE 4.-The detection of the MspI RFLPs and their association with various agouti locus alleles. A Southern blot is shown of MspI digested DNAs which were electrophoresed in a 1.5% agarose gel and hybridized with radiolabeled pP0.5. At the left the positions of the 1.7-kb (A type) fragment and 1.2-kb (B type) fragment are shown. The lower fragment in the righthand track is the 1.0-kb (C type) fragment. The a/a strain used was C57BL/6J, and, from left to right, the A"/A" strains used were 129/Sv and 129/ReJ. The A/ A strain used was C3H/HeJ. All other strains used are unambigu- ously listed in Table 1.

1.0 kb (the C configuration) in the DNA from mice heterozygous for the AY allele. This shorter fragment results from the presence of an MspI site in the Emv- 25 proviral LTR and comprises the 5' viral-host junction from Emv-I5 which is detected by pP0.5. This MspI site was mapped by hybridization of pP0.5 to double digests of C57BL/6J-AJ/a DNA and its presence was confirmed by DNA sequencing of the Emv- 15 LTR (data not shown).

Table 1 summarizes the RFLPs we have detected in the various strains we have tested. A clear pattern can be discerned from these data, with some important exceptions which will be discussed below. The A pattern of RFLPs is generally specific to the wild type (A) allele and to mutations which were derived from A. The B pattern of RFLPs is generally specific to mice carrying the nonagouti (a) allele and alleles (A", A', and A"') which were derived by mutation from nonagouti (DICKIE 1969a,b). In addition, the black and tan allele (af), which is an old mutant of the mouse fancy (DUNN 1928), also carries this configuration. Our data would indicate that this mutation also probably arose from a nonugouti stock. This interpretation is supported by the observation that mutations from a to a' are relatively common (GREEN 198 1). With the exception of the SM/J strain, all of the inbred strains we have tested that carry the nonagouti allele retain the B pattern. Interestingly, the SM/J strain has been maintained for greater than 80 inbred generations by mating mice heterozygous at the agouti locus (A"/u) against mice homozygous for nonagouti (ala) (FESTING

Markers for the Agouti Locus 75 1

TABLE 1

Summary of the RFLPs present in various mouse strains carrying different agouti locus alleles

Strain" Allele' HindIII' MspI'

C57BL16J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J C57BL/6J 129lSv 129lSv 12915 129/ReJ B ALB/cJ C3H/HeJ CBAIJ SJLIJ SWRIJ MWT/Le

RFIJ PIJ NZB/Bl NJ C57L/J DBA/ 1 J BUB/BnJ AKRIJ AIJ AIHeJ YBR/Ki YBR/Ki SWJ SWJ AEJ/Gn

ala A '/a Aq/AY Ai/. Aq/a A"J/A"J A'/A' Ay/A" A"/A" A"/A" A"/A" AIA AIA AIA A/A AIA atla' a '/a a"/a A' AwlAS A" T26H a6H/T26H a6H T30H A/+ a ala a la ala a la a la a la a la a la a la

ala a la

AY/a

Aula a*/ae

B

A

B B B A A A A A A A A A B A AIB A A AIB B B B B B B B B B B B A A A

AIB

A D

B

A

B B B

A A A A A A A A B A

A A

B B B B B B B B B B B A A A

B/C

AIB

AIC

A/B

A D

a The inbred genetic background in which the various alleles are maintained is listed where known.

* The As mutation, as is noted in the text, is a position effect mutation associated with an inversion and is not allelic with the other listed mutations. ' The A pattern of RFLPs represents the absence of an MspI site

and the presence of a Hind111 site as is shown in Figure 2B. The B pattern of RFLPs represents the presence of an MspI site and the absence of a Hind111 site as is shown in Figure 2C. The C MspI polymorphism represents the presence of a 1 .O-kb pP0.5-homologous fragment which is a host-viral junction fragment (see text for details).

1979). Since black (a la) SM/J mice now contain the A pattern of RFLPs, it would appear that a recombination event has occurred, transferring the A pattern of RFLPs from linkage with the A" allele to linkage with the a allele, a change which has subsequently become inbred to homozygosity in this small breeding colony.

The U' mutation: As has been mentioned above, the lethal yellow (AY) allele and the radiation-induced nonugouti lethal (a') allele are prenatal homozygous lethals which fail to complement in heterozygous com-

bination (AY/a') (LYON, FISHER and GLENISTER 1985). It has been suggested, for this and other reasons, that the al allele may be a deletion. It is interesting to note in this regard that aL/u mice carry both the A and B RFLPs (see Figure 4), indicating that the U' mutation does not delete the DNA sequences immediately surrounding the proviral integration site.

The AY mutation and a recombinant which lacks Emv-15: Mice heterozygous for the AY allele on the C57BL/6J or 129/Sv genetic backgrounds contain the A pattern of HindIII polymorphism with the pRI probe. Although the AY allele is an old mutant of the mouse fancy and its derivation is unknown, the presence of the A HindIII site polymorphism in these two strains argues that it originally arose from an allele other than the nonugouti allele. These two strains also contain the C MspI polymorphism (with the pP0.5 probe) which results from the presence of the Emu-Z5 provirus. However, one important exception to the latter correlation is the AY allele carried on the YBR/ Ki strain. This allele has been carried congenically and heterozygously for 130 inbred generations (Dr. M. OHMORI, personal communication). YBR/Ki-AY/u mice are yellow in coat color but lack the Emu-15 proviral integration. This was determined in two ways: (1) Southern blots conducted on YBR/Ki-AY/a DNA using the ecotropic virus-specific subclone pECenv (CHAN et al. 1980) failed to detect the Emu-Z5 provirus; and (2) Southern blots on the same DNAs using both pRI and pP0.5 failed to detect any difference in the mobility of fragments generated from this strain compared to DNA from a mouse homozygous for the nonagouti allele (ala). An example of the latter experiment is shown in Figure 5 . In this case, the DNAs were digested with MspI and were probed with pP0.5. YBR/Ki-AY/a DNA does not show the C polymorphism characteristic of the presence of the provirus.

The loss of the Emu-Z5 provirus from this strain may have occurred through viral excision or through recombination. The data shown in Figure 5 favor a recombinational mechanism for two reasons. First, the retention of one proviral LTR within the AY/a DNA (which has been shown to occur in at least some cases upon proviral excision [VARMUS, QUINTRELL and O R - TIZ 198 1 ; COPELAND, HUTCHISON and JENKINS 19831) would be easily detectable by an alteration in fragment mobilities in Figure 5, since the detected fragments span the proviral integration site. Second, kBR/Ki-AY/a DNA is now homozygous for the B pattern of polymorphisms suggesting that recombination has occurred within this region of chromosome 2 with the consequent exchange of polymorphisms. Although it is at present unclear if YBR/Ki-AY/a mice retain all the pleiotropic phenotypic features associated with C57BLIGJ-AYIa mice, our findings strongly suggest that the Emu-Z5 provirus per se is not involved

752 M. Lovett et al.

A A A A

C3H;ieJ A SWWJ A BALB/cJ A sJL1J A

TABLE 2

Strain distribution pattern of RFLPs in recombinant inbred strains

"

CXB-D A A A A CXB-E

CXB-G A A CXB-H B B

+ 1.7

c1.2

FIGURE 5.-The YBR-A'/a strain of mice lacks the Emv-Z5 provirus. A Southern blot is shown of MspI digested DNAs which were electrophoresed in a 1.5% agarose gel and hybridized with radiolabeled pP0.5. DNAs from both YBR-A'/a and YBR-a/a mice contain only the 1.2-kb (B type) fragment which spans the proviral integration site (see Figure 2C). T h e A/A strains used in this experiment were, from left to right, C3H/HeJ and SWR/J.

BxH-19 14 12 1 1 10 9 8 7 6 4 3 2

5.4 kb-

2.6 kb-

FIGURE 6.-The strain distribution pattern of the HindIII RFLP in BXH RI strains. A Southern blot is shown of Hind111 digested DNAs from the BXH RI strains (numbered a t the top of the figure), and from the two progenitor strains (in the righthand two tracks), which were electrophoresed in a 0.8% agarose gel and were hybridized with radiolabeled pRI. T h e positions of the 2.6-kb (A type) fragment and the 5.4-kb (B type) fragment a re shown at the left.

in the production of the yellow coat color. Genetic mapping: Since the only evidence that

suggests that the Emu-15 provirus is genetically close to the agouti locus is its retention in three inbred straihs which carry AY, we sought to gain a more quantitative assessment of the degree of linkage between these two markers by using the RFLPs described above and a panel of recombinant inbred (RI) mouse strains (BAILEY 197 1) commercially available from The Jackson Laboratory. The progenitors of each of these RI strains differed at the agouti locus, with one progenitor being wild type (A/A) and the other nonagouti (ala). DNAs from these RI strains were separately digested with MspI and HindIII and Southern blots of these were hybridized with radiolabeled pP0.5. A representative blot of the BXH series of RI strains is shown in Figure 6, and a complete listing of the strain distribution pattern (SDP) is shown in Table 2. In all cases, the polymorphic pattern is completely concordant with the agouti locus (Dr. B. TAYLOR, personal communication), with 95% confi-

Strain Hind111 Mspl

C57BL/6J B B C57LI1 B B

Strain Hind111 Msgl

BXJ- 1 B B BXI-2 B B

BXH-2 B B BXH-3 A A BXH-4 B B BXH-6 BXH-7 BXH-8 BXH-9 BXH-IO BXH-11 BXH-12 BXH-14

A A B B A A A A B B B B A A A A

BXH-19 B B l

CXB-I B B

CXB-K B B SWXL-4 A A SWXL-7 B B SWXL-12 B B SWXL-14 B B SWXL-15 B B SWXL-16 A A SWXL-17 B B

CXB-J B B

Progenitor strains are listed first, followed by the various RI strains. See materials and methods for details on the progenitor strains for each set of RI strains. ' T h e A and B RFLP patterns are used here as detailed in the footnotes to Table 1 and in Figure 2, B and C.

dence limits of 0.00 to 3.79 cm and 99% confidence limits of 0.00 to 5.81 cm (SILVER 1985).

Physical mapping: The two reciprocal chromosomal translocations (n2;8]26H and q 2 ; I1130H) which affect agouti locus function and the As inversion all have cytologically identifiable breakpoints within band H I of chromosome 2, and the physical location of the agouti locus has therefore been defined as being within this band. To determine whether our cloned DNA sequences confirm this location, we conducted in situ hybridizations onto metaphase spreads of mouse chromosomes using the 3H-labeled subclone pRI (CHENG et al. 1986). Because the normal mouse karyotype contains only acrocentric chromosomes and since we had already determined that the sequences within pRI are located on chromosome 2 by hybridization to a panel of mouse X hamster somatic cell hybrid DNAs (data not shown), we utilized metacentric Robertsonian translocation chromosomes which contain chromosome 2. The presence of two R6(2. I7) l lRma metacentric chromosomes in each spread allowed easy identification to be made even without G-banding. Analysis of 175 unbanded meta- phases showed that 54 chromosome spreads (30.8%) had hybridization events (grains) on a translocated chromosome 2 and that 17.6% (6 1 of 346) of all grains were located on chromosome 2. All chromosome spreads on which there were positive hybridization events on the marker chromosome 2 were photo- graphed, and the relative length of this chromosome 2 and position of the grains on it were measured. The

Markers for the Agouti Locus 753

N 0.1 5

0 g 0.2

2 0.3

I 0.4 0

8 0.5 I I- a 0.6 z ‘ 0.7 w 1 4 Oe8 E 0.9

1 .o

b

c

5 10 15 20

GRAINS FIGURE 7.-Physical location of pRI on mouse chromosome 2.

(Left) Distribution of silver grains by relative distance from centro- mere to telomere on 61 unbanded chromosomes 2 contained in Robersonian fusion chromosomes. (Right) Distribution of silver grains on 44 G-banded chromosomes 2.

distribution of grains by length on unbanded spreads is shown on the left side of Figure 7. Of the 281 G- banded spreads examined, 13.2% (37 of 281) had hybridization events on chromosome 2 and 20% (44 of 220) of grains hybridized to either (or very occa- sionally both) of the two chromosomes 2. Of these, 27.3% (12 of 44) were on band H1 as is shown on a karyogram of chromosome 2 (Figure 7), directly confirming the previously assigned physical map location.

DISCUSSION

The experiments described in this report were performed with the aim of obtaining a more quantitative assessment of the degree of genetic linkage between the Emu-15 provirus and the functional agouti locus of the mouse. The previously described association of Emu-15 with the AY mutation in three inbred strains in which it is carried congenically and heterozygously itself implies a close genetic linkage. We have here further defined this linkage in two ways. First, we have shown that two RFLPs within the mouse DNA close to the Emu-15 preinsertion site are associated with the nonagouti allele of the agouti locus and mutations derived from it in a large number of inbred mouse strains. Second, using these RFLPs and 28 recombinant inbred mouse strains we have shown that these molecular markers are concordant within the limits of resolution of this technique. We have also

physically mapped these sequences by in situ hybridization. These data indicate that our molecular probes are derived from band H1 of chromosome 2, thus correlating the genetic and physical location of the agouti locus and directly confirming the previously assigned map location.

While our genetic mapping data indicate that the Emu-15 provirus and the agouti locus are very closely linked, our finding that YBR/Ki-AY/a mice lack the Emu-1 5 provirus indicates that Emu-15 probably does not play a direct part in the causation of the AY- associated yellow coat color. One possible explanation for this observation is that the Emu-15 proviral integration was superimposed upon the AY mutation in the course of inbreeding and that the allele carried in the YBR/Ki strain was never associated with Emu-1 5 . However, our data suggest that a recombination event, which might have led to the loss of Emu-15, did occur in this strain to transfer the B HindIII RFLP to linkage with AY, which in other strains is associated with the A HindIII RFLP. A detailed comparison between the phenotypic features associated with the AY mutation on the YBR/Ki and C57BL/6J genetic backgrounds should resolve whether the provirus and the apparently altered transcription unit which flanks it affect the other AY-associated phenotypic features.

The limits of resolution in genetic mapping with RI strains are unfortunately rather imprecise (SILVER 1985). To refine further the linkage between our markers and the agouti locus to within a fraction of a centimorgan would require an extensive linkage analysis on many hundreds of progeny from a cross of mice heterozygous for the RFLPs we have described. Our data suggest that an appropriate cross for such an analysis would be a heterozygous combination of the wild-type agouti allele and the aforementioned black and tan allele (Alat X Ala‘). These alleles differ in their RFLP patterns and a cross of this type has the added advantage that all three types of progeny (A/A, Ala‘ and a f / a f ) have different coat colors, thereby allowing all progeny to be scored for a potential recombination event. However, it is possible that an extended “chromosome walk,” using the molecular probes we have isolated as a starting point, and correlation with the available chromosomal translocations and radiation-induced mutations at the agouti locus, will define the exact location of the functional agouti locus before such a time consuming analysis can be completed.

We thank TEOD~SIA ZAMORA for excellent technical assistance. This work was supported by grant HD-03132 from the National Institute of Child Health and Human Development. M. L. was supported in part by the Weingart Foundation Program in Devel- opmental Genetics.

754 M. Lovet t et al.

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Molecular Markers for the agouti Coat Color ... - Genetics · Molecular Markers for the agouti Coat Color Locus of the Mouse Michael Lovett, Zai-yu Cheng,' Estrella M. Lamela, Tohru