Journal of Heredity Advance Access originally published online on January 13, 2005
Journal of Heredity 2005 96(2):145-149; doi:10.1093/jhered/esi022
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Brief Communication |
A Nucleotide Substitution Responsible for the Tawny Coat Color Mutation Carried by the MSKR Inbred Strain of Mice
From the Laboratory Animal Facility, Research Center for Medical Sciences, Jikei University School of Medicine, Tokyo, Japan (Wada and Ohkawa); Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan (Kunieda); Institute for Laboratory Animal Research, Graduate School of Medicine, Nagoya University, Aichi, Japan (Nishimura); Port of Nagoya Public Aquarium, Aichi, Japan (Kakizoe-Ishida); Department of Tropical Medicine, Jikei University School of Medicine, Tokyo, Japan (Watanabe); and Laboratory of Animal Breeding and Genetics, Graduate School of Biosphere Science, Hiroshima University, Hiroshima, Japan (Tsudzuki)
Address correspondence to A. Wada, Laboratory Animal Facility, Research Center for Medical Sciences, Jikei University School of Medicine, Nishi-Shinbashi 3-19-18, Minatoku, Tokyo, Japan 105-8461, or e-mail: adumiw{at}jikei.ac.jp.
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Abstract |
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"Tawny" is an autosomal recessive coat color mutation found in a wild population of Mus musculus molossinus. The inbred strain MSKR carries the mutation. The causative gene Mc1rtaw of the tawny phenotype is the second recessive allele at the melanocortin 1 receptor locus and is dominant to the first recessive allele, "recessive yellow" (Mc1re). The Mc1rtaw gene has six nucleotide substitutions, and its forecasted transcript has three amino acid substitutions (i.e., V101A, V216A, W252C). Though the nucleotide substitutions leading to V101A and V216A exist in various mouse strains, the nucleotide substitution leading to W252C exists in only tawny-colored mice. Thus this substitution is considered to be responsible for the expression of the tawny coat color. The frequency of the allele having this nucleotide substitution was 9.21% in the wild M. m. molossinus population inhabiting Sakai City, Osaka Prefecture, Japan, where the ancestral mice of the MSKR strain were captured.
The extension locus (E, now Mc1r) controls coat or feather color variants in animals. Pigmentation mutants resulting from the mutation of this locus have been found in several animals, for example, cattle, chicken, dog, fox, horse, and mouse (Adalsteinsson et al. 1995; Andersson and Sandberg 1982; Silvers 1979; Sponenberg and Bigelow 1987; Vage et al. 1997). In mammals, dominant alleles at this locus generally extend the black area of individual hair shafts (Doolittle et al. 1996). In 1993, Robbins et al. (1993) reported the nucleotide sequence of the mouse
-melanocyte stimulating hormone (-MSH) receptor (now melanocortin 1 receptor, Mc1r) gene with four mutant alleles at the extension locus and proved that the extension locus encodes this receptor. The MC1R is a G protein coupled receptor consisting of seven transmembrane domains. Binding of -MSH to its receptor stimulates melanocytes to synthesize cyclic adenosine monophosphate (cAMP) by signal transduction via G protein, and consequently the melanocytes produce black pigment (eumelanin) (Robbins et al. 1993). After the research of Robbins et al. (1993), nucleotide and amino acid sequences responsible for the melanocortin 1 receptor were reported in various kinds of animals, for example, cattle, horse, pig, dog, fox, guinea pig, and mouse (Adalsteinsson et al. 1995; Cone et al. 1996; Kijas et al. 1998; Lu et al. 1994; Mariani et al. 1996; Marklund et al. 1996; Newton et al. 2000; Vage et al. 1997; Valverde et al. 1995, 1996). At the Mc1r locus of mice, three dominant mutant alleles (sombre, sombre-3J, and tobacco darkening) and one recessive mutant allele (recessive yellow) have been reported (Doolittle et al. 1996). Both sombre and sombre-3J mice have an entirely black coat, while the tobacco darkening mouse shows black fur in the dorsal region and agouti patterned fur on the flanks. The recessive yellow mouse has no black area on the individual hair shafts and shows a yellow coat everywhere over the body, except for a few black hairs appearing in juvenile mice (Doolittle et al. 1996; Robbins et al. 1993).
In 1999 we reported a new mutation, "tawny," at the Mc1r locus that was found in Japanese wild mice (Mus musculus molossinus). The tawny (Mc1rtaw) mutation is a recessive allele dominant over the recessive yellow (Mc1re) allele. The tawny mouse shows light yellowish brown on the dorsal region with a white belly and black eyes (Figure 1). The dorsal hair shows so-called agouti pattern, consisting of a greatly lengthened subapical yellow region and reduced black region (Wada et al. 1999). The Mc1rtaw mutation is maintained in the MSKR strain (Wada et al. 2000), its cognate MSKQ strain, and the Mmsw line.
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In this article we researched nucleotide substitutions of the Mc1r gene in the tawny mutant (MSKR strain) and 24 other inbred strains of mice, along with mice from wild populations of M. m. molossinus, using nucleotide sequencing and/or the restriction fragment length polymorphism (RFLP) technique.
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Materials and Methods |
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Animals
The MSKR strain of mice (fixed for the tawny coat color mutation, n = 4) and 24 other inbred-strain mice were used. The 24 strains, which show nontawny coat colors, were A/J (3), AEJ/GnLe (2), BALB/cA (4), BFM/2 (2), C3H/HeJ-Mc1rsom/Mc1rsom (2), C3H/HeN (4), C57BL/6J-Mc1re/Mc1re (2), C57BL/6N (4), CASA/Rk (2), CAST/EiJ (2), CBA/N (3), DBA/2N (2), DDK/Nga (3), IS/Cam (1), MMNF (2), MOM (3), MSKA (4), MSKD (2), MSKM (2), MSKO (2), MSKZ (4), MSM/Msf (3), NC/Nga (4), and SM/J (3). The numeral in parentheses indicates the number of mice tested. All of these strains have been maintained in Osaka Prefecture University except the A/J, BFM, CASA, CAST, IS, MSM, and SM/J strains. The A/J, MSM, and SM/J strains have been maintained in the Institute for Laboratory Animal Research, Graduate School of Medicine, Nagoya University, Japan. The BFM/2, CASA, and CAST strains were kindly provided by the National Institute of Genetics (Shizuoka, Japan) and the IS strain was kindly provided by Wakayama Medical University (Wakayama, Japan). BFM/2 was derived from M. m. brevirostris, CASA and CAST were derived from M. m. castaneus, IS was established from the hybrid of M. m. musculus and M. m. praetextus. MMNF, MOM, MSKA, MSKD, MSKM, MSKO, MSKR, MSKZ, and MSM were established from M. m. molossinus.
In addition to these strains, we analyzed 105 wild mice (M. m. molossinus) captured in the Kinki-Shikoku area of Japan during the period from January 1991 to May 1992. Of the 105 mice, 23 were captured in Minoh City, Osaka Prefecture; 38 each were captured at the northern and southern parts of Sakai City, Osaka Prefecture; and 6 were captured in Wakimachi, Tokushima Prefecture (Figure 2).
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Nucleotide Sequencing
Standard polymerase chain reaction (PCR) was performed with genomic DNA of MSKA, MSKM, MSKR, and MSM inbred mice using the GeneAMP 9700 PCR system (PerkinElmer, Wellesley, MA). A sense primer "mMc1r-1-for" (5'-TCTGAGGGATGTCAGAGACCC-3') and an antisense primer "mMc1r-2-rev" (5'-GCAGTCACAGTTACCCT TTCTCC-3') were originally designed based on the nucleotide sequence (accession no. X65635) reported by Mountjoy et al. (1992). The mMc1r-1-for and mMc1r-2-rev primers amplify a 1229 bp fragment including the entire coding region of the mouse Mc1r gene. PCR amplification was carried out under the following conditions: one cycle consisting of denaturation at 94°C for 5 min, 40 cycles consisting of denaturation at 94°C for 30 s, annealing at 61°C for 45 s, and extension at 72°C for 60 s in a reaction mixture containing 0.5 mM of each primer, 0.05 U/ml of Taq polymerase, 0.2 mM each of dNTPs, and 1.5 mM MgCl2. The amplified fragments were purified by polyethylene glycol 6000 and cycle sequenced using the Dye Terminator Cycle Sequencing FS Ready Reaction Kit with the primers described above and additionally designed primers "mMc1r-2-for" (5'-CTCCATCTTCTATGCGCTGC-3') and mMc1r-1-rev (5'-GAAAGTGACGAGGCAGAGCAG-3'), based on the manufacturer's instructions. The reactants were sequenced using an ABI model 373 automated DNA sequencer (Applied Biosystems, Foster City, CA).
PCR-RFLP Analysis
In order to reveal the relationship between the nucleotide substitutions and the tawny phenotype, RFLPs of the Mc1r gene were compared between mice of the tawny mutation strain MSKR and the other 24 inbred strains. In addition, the incidence of RFLP identical to the tawny was investigated in wild populations of M. m. molossinus in the Kinki-Shikoku area of Japan. A 986 bp PCR product, which includes the entire 945 bp coding region of the mouse Mc1r gene, was amplified by a set of Mc1r-specific PCR primers described by Robbins et al. (1993). PCR amplification was carried out with the same methods mentioned above. In this experiment, however, annealing temperature was adjusted at 65°C and purification of the PCR products was carried out with ethanol. The purified PCR products were digested with endonucleases ApaI, Cac8I, and EcoRII, electrophoresed on 0.6% agarose gels, and visualized by ethidium bromide staining.
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Results |
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Nucleotide Sequencing
Table 1 shows the nucleotide substitutions observed in the Mc1r alleles of four inbred strains of molossinus mice in comparison with the registered nucleotide sequence in GenBank (accession no. X65635, which was identified based on the Cloudman S91 melanoma cell line separated from a hybrid of BALB/cJ and DBA), along with their forecasted amino acids. Five nucleotide substitutions were identified at the positions listed; that is, substitution I (51T ... C), II (302T ... C), III (606G ... A), IV (647T ... C and 648C ... T), and V (756G ... T). The first four were observed both in wild-type and tawny-colored strains. The substitution V, however, was observed only in the MSKR strain that has the "tawny" coat color. According to the amino acid code, nucleotide substitutions I (AAT ... AAC) and III (GCG ... GCA) are silent. However, substitutions II, IV, and V cause amino acid substitutions; that is, 101 valine to alanine, 216 valine to alanine, and 252 tryptophan to cysteine, respectively (Table 1).
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PCR-RFLP Analysis
The nucleotide substitutions II, III, IV, and V lead to recognition sites of endonucleases Cac8I, HinfI, EcoRII, and ApaI, respectively (Table 1). Thus we investigated the distribution of these recognition sites across various mouse strains derived from some subspecies, the results of which are summarized in Table 2. The Cac8I recognition site was observed in all strains of M. m. molossinus. Other strains derived from other subspecies had or did not have the recognition site. The presence or absence of the HinfI recognition site clearly distinguished the strains of M. m. molossinus from the strains of other subspecies; that is, the site was missing in the strains derived from M. m. molossinus. The EcoRII recognition site existed in all 25 strains investigated. The ApaI recognition site was missing in only the MSKR strain of molossinus mice that has the tawny (Mc1rtaw/Mc1rtaw) coat color.
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In addition to the strains above, we performed RFLP analysis for wild mice. Among 38 wild mice captured in the southern part of Sakai City, where the original tawny mutant had been captured, 5 mice had no recognition site of ApaI—2 were homozygotes and 3 were heterozygotes—that is, the allele frequency of Mc1rtaw was 9.21% in this population (Figure 2). The two homozygotes showed tawny coat color and the others (three heterozygotes) showed wild-type coat color. On the other hand, all 67 mice captured at other places had the recognition site for ApaI in their Mc1r genes.
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Discussion |
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In the tawny mutant, we found six nucleotide substitutions classifiable to five types (substitutions I–V in Table 1) at the Mc1r locus. The nucleotide substitutions at base pair positions 51 and 606 (substitutions I and III) are silent. Thus these two are not thought to be the cause of the tawny mutation. The nucleotide substitution at base pair 302 (substitution II) led to an amino acid substitution (Table 1). However, this substitution was also found in wild-type molossinus mice and other strains derived from other subspecies (Tables 1 and 2). Thus the nucleotide substitution at 302 is thought not to be the cause of the tawny coat color.
As shown in substitution IV of Table 1, the nucleotides 647 and 648 of the mouse Mc1r gene reported by Mountjoy et al. (1992) were T and C, respectively, while those of our results from molossinus strains were C and T, forecasting alanine at 216. Nucleotide 648 results in the recognition site of EcoRII (Table 1). In addition to molossinus mice, all 24 strains derived from other subspecies also had no recognition site for EcoRII (Table 2). This result suggests that no mice have a nucleotide sequence identical to that reported by Mountjoy et al. (1992) at the position. Furthermore, the alanine at 216 has been found in many kinds of mammals that have different phenotypes from the tawny mouse (Adalsteinsson et al. 1995; Cone et al. 1996; Kijas et al. 1998; Lu et al. 1994; Mariani et al. 1996; Marklund et al. 1996; Newton et al. 2000; Vage et al. 1997; Valverde et al. 1995, 1996). Thus the nucleotide substitutions at 647 and 648 are thought not to be responsible for the tawny coat color.
Although substitutions I–III described above were observed both in wild-type and tawny-colored mice, substitution V (756 guanine to thymine), which leads to the 252 tryptophan to cysteine substitution, was observed only in tawny-colored mice (Table 1). This result strongly suggests that this substitution results in the tawny phenotype. Amino acid 252 is conserved as tryptophan in all reported animals (Klungland et al. 1995; Marklund et al. 1996; Mountjoy et al. 1992; Robbins et al. 1993; Vage et al. 1997; Valverde et al. 1995). Amino acid 252 is involved in the sixth transmembrane domain of MC1R (Mountjoy et al. 1992; Robbins et al. 1993). The replacement of a hydrophobic amino acid residue (tryptophan) with a hydrophilic one (cysteine) would change the -helix structure of the transmembrane domain of MC1R. Moreover, the sixth transmembrane domain forms an -MSH binding pocket with first and third transmembrane domains (Prusis et al. 1995). In site-specific mutant research using recombinant COS7 cells (from African green monkey), replacement of an amino acid residue in the sixth domain has been reported to decrease -MSH binding affinity (Frandberg et al. 1994). The tawny-type MC1R with W252C also may decrease -MSH binding affinity. The altered affinity of MC1R to its ligand might lead to extension of yellow color at the subapical region of the hair shaft. Another possibility is that a decrease in constitutive activity and/or stability of the MC1R protein might result in the tawny phenotype. A ligand binding affinity test will solve this problem.
The allele frequency of Mc1rtaw was 9.21% in the wild molossinus mice captured in the southern part of Sakai City, Osaka Prefecture, Japan (Figure 2), where the original tawny mutant had been captured. The Mc1rtaw mutation is thought to have originally occurred in the population at the southern part of Sakai City, because the allele frequency of Mc1rtaw was 0% in other areas adjacent to the southern part of the city.
The amino acid substitution W252C is quite likely to be the cause of the tawny coat color, as mentioned. However, there remains a possibility that some other abnormality occurring at or around the Mc1r coding region might give rise to the mutant color.
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Acknowledgments |
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We thank Dr. K. Moriwaki (National Institute of Genetics, Shizuoka, Japan) and Dr. M. Miyajima (Wakayama Medical College, Wakayama, Japan) for their kind impartation of wild-derived inbred mice. This research was partially supported by a Grant-in-Aid for JSPS Fellows (no. 00932) from the Ministry of Education, Science, Sports, and Culture, Japan.
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Footnotes |
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Corresponding Editor: Muriel Davisson
Received March 30, 2004
Accepted August 20, 2004
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References |
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