Genetic Organization of the agouti Region of the Mouse

The agouti locus on mouse chromosome 2 acts via the hair follicle to control the melanic type and distribution of hair pigments. The diverse phenotypes associated with various agouti mutations have led to speculation about the organization of the agouti locus. Earlier studies indicated that two presumed agouti alleles, lethal yellow (Ay) and lethal light-bellied nonagouti (ax), are pseudoallelic. We present genetic data showing probable recombination between Ay and three agouti mutations (at, a, and ax), which suggest that Ay is a pseudoallele of the agouti locus. The close linkage of an endogenous ecotropic murine leukemia provirus, Emv-15, to Ay provides a molecular access to genes at or near the agouti locus. However, previous studies suggested that the Emv-15 locus can recombine with some agouti alleles and therefore we analyzed mice from recombinant inbred strains and backcrosses to measure the genetic distance between various agouti alleles and the Emv-15 locus. Our data indicate that the Emv-15 locus is less than 0.3 cM from the agouti locus. These experiments provide a conceptual framework for initiating chromosome walking experiments designed to retrieve sequences from the agouti locus and give new insight into the genetic organization of the agouti region.     

Allelic Variation within the Emv-15 Locus Defines Genomic Sequences Closely Linked to the agouti Locus on Mouse Chromosome 2

Gene(s) at the agouti locus act within the microenvironment of the hair follicle to switch pigment synthesis in the melanocyte between eumelanin (black or brown pigment) and phaeomelanin (yellow pigment). Many phenotypic variants of this locus have been described. The mechanism(s) of gene action causing such variation in coat-color phenotype is not known. The close linkage of an endogenous ecotropic murine leukemia provirus, Emv-15, to the lethal yellow mutation of the agouti locus provides a means to molecularly access genes at or near the agouti locus. We have identified and used a unique mouse sequence flanking the Emv-15 provirus to define three alleles of the Emv-15 locus. We found a correlation between the presence of specific Emv-15 alleles and the origins of specific agouti locus mutations, confirming close linkage. However, we found some exceptions which suggest that the Emv-15 locus is closely linked to, but genetically separable from, the agouti locus.     

Chromosomal assignment of two endogenous ecotropic murine leukemia virus proviruses of the AKR/J mouse strain.

The AKR/J mouse strain is genetically fixed for three different ecotropic murine leukemia virus genomes, designated Akv-1, Akv-3, and Akv-4 (Emv-11, Emv-13, and Emv-14). With recombinant inbred strains and crosses with linkage-testing stocks, Akv-3 and Akv-4 were placed on the mouse chromosome map. Akv-3, which encodes a replication-defective provirus, maps near the agouti coat color locus, a, on chromosome 2. Akv-4, which is replication competent, maps near the neurological mutant gene locus trembler, Tr, on chromosome 11. Akv-1 and Akv-2 (Emv-12), an ecotropic provirus carried by AKR/N but not AKR/J, have previously been mapped to chromosome 7 and 16, respectively. Thus, the four Akv proviruses mapped to date are on four different chromosomes. Akv-3 is the second ecotropic murine leukemia virus provirus to be mapped near the agouti locus. The results are discussed in relation to possible nonrandomness of viral integration.     

Recombinant Inbred Strain and Interspecific Backcross Analysis of Molecular Markers Flanking the Murine Agouti Coat Color Locus

Recombinant inbred strain and interspecific backcross mice were used to create a molecular genetic linkage map of the distal portion of mouse chromosome 2. The orientation and distance of the Ada, Emv-13, Emv-15, Hck-1, Il-1a, Pck-1, Psp, Src-1 and Svp-1 loci from the beta 2-microglobulin locus and the agouti locus were established. Our mapping results have provided the identification of molecular markers both proximal and distal to the agouti locus. The recombinants obtained provide valuable resources for determining the direction of chromosome walking experiments designed to clone sequences at the agouti locus. Comparisons between the mouse and human genome maps suggest that the human homolog of the agouti locus resides on human chromosome 20q. Three loci not present on mouse chromosome 2 were also identified and were provisionally named Psp-2, Hck-2 and Hck-3. The Psp-2 locus maps to mouse chromosome 14. The Hck-2 locus maps near the centromere of mouse chromosome 4 and may identify the Lyn locus. The Hck-3 locus maps near the distal end of mouse chromosome 4 and may identify the Lck locus.     

The long-range restriction map surrounding the mouse agouti locus reveals a disparity between physical and genetic distances

Isolation of a gene based on its location, which depends on aligning physical landmarks with the genetic map, can yield basic information about genome structure and organization. As a first step toward isolating the mouse agouti (A) locus, we have begun to define the physical position of this gene relative to genetically linked DNA probes from the Psp, Emv-15, and Src loci. Using a combination of pulsed-field gel techniques that include partial digestion with rare-cutting restriction enzymes and analysis of polymorphic sites present in certain inbred strains, we have constructed long-range restriction maps for each of the probes that span a total of more than 3000 kb. The Src and Emv-15 probes are less than 600 kb apart, but are separated from the Psp probe by at least 1500 kb. By determining the position of a 75-kb deletion that inactivates agouti function, we have localized the A locus to within 500 kb of the Psp probe, but more than 600 kb away from the Emv-15 probe. These physical distances contrast with the known recombination frequencies, 3 +/- 3 cM for A-Psp and less than 0.3 cM for A-Emv-15, and suggest that recombination between A and Emv-15 may be suppressed.     

Molecular characterization of the mouse agouti locus.

The agouti (a) locus acts within the microenvironment of the hair follicle to regulate coat color pigmentation in the mouse. We have characterized a gene encoding a novel 131 amino acid protein that we propose is the one gene associated with the agouti locus. This gene is normally expressed in a manner consistent with a locus function, and, more importantly, its structure and expression are affected by a number of representative alleles in the agouti dominance hierarchy. In addition, we found that the pleiotropic effects associated with the lethal yellow (Ay) mutation, which include pronounced obesity, diabetes, and the development of neoplasms, are accompanied by deregulated overexpression of the agouti gene in numerous tissues of the adult animal.     

Association of Xmv-10 and the non-agouti (a) mutation explained by close linkage instead of causality

In a previous survey of endogenous proviruses among inbred mouse strains, the Xmv-10 provirus was found only in strains that carried the non-agouti (a) mutation (Frankel et al. J. Virol. 63: 1763–1774, 1989). To determine whether insertion of Xmv-10 caused the a mutation, we cloned a portion of Xmv-10 and its insertion site. Using a fragment of flanking cellular DNA as a Southern hybridization probe, we found that the Xmv-10 provirus was still present in revertant alleles of a to a ^tor A W.A restriction fragment length variant (RFLV) in cellular DNA at the Xmv-10 insertion site was found to be correlated with the presence or absence of the provirus among inbred strains of laboratory mice regardless of their agouti allele. This correlation did not extend to wild mice, however, in which none of the samples contained Xmv-10, yet one, Mus domesticus poschiavinus, contained the insertion site RFLV correlated with Xmv-10 in laboratory mice. Analysis of an intersubspecific backcross with RFLVs at the insertion sites of Xmv-10 and Emv-15 (an endogenous provirus associated with A ^y)revealed the following genetic map information: cen-A-0.31±0.31 cM-Emv-15-0.62±0.27 cM-Xmv-10-tel. Haplotype analysis of inbred strains in which a was not associated with Xmv-10 and in which A ^ywas not associated with Emv-15 demonstrated that these exceptions were explained most simply by a single recombination that disturbed the linkage relationships evident in most inbred strains. These results demonstrate that Xmv-10 did not cause the a mutation, suggest that insertion of Xmv-10 occurred recently in the evolution of laboratory mice, and show that the associations between agouti alleles and endogenous proviruses are due to linkage disequilibrium.     

Physical and Genetic Characterization of a 75-Kilobase Deletion Associated with A(l), a Recessive Lethal Allele at the Mouse Agouti Locus

The agouti locus (A) of the mouse determines the timing and type of pigment deposition in the growing hair bulb, and several alleles at this locus are lethal when homozygous. Apparent instances of intragenic recombination and complementation between different recessive lethal alleles have suggested that the locus has a complex structure. We have begun to investigate the molecular basis of agouti gene action and recessive lethality by using a series of genetically linked DNA probes and pulsed field gel electrophoresis to detect structural alterations in radiation-induced agouti mutations. Hybridization probes from the Src and Emv-15 loci do not reveal molecular alterations in DNA corresponding to the ae, ax, and al alleles, but a probe from the parotid secretory protein gene (Psp) detects a 75-kilobase (kb) deletion in DNA containing the non-agouti lethal allele (al). The deletion is defined by a 75-kb reduction in the size of BssHII, NotI, NruI and SacII high molecular weight restriction fragments detected with the Psp probe and is located between 25 kb and 575 kb from Psp coding sequences. Because the genetic distance between A and Emv-15 is much less than A and Psp, there may be a preferred site of recombination close to Psp, or suppression of recombination between A and Emv-15. The al deletion has allowed us to determine the genotype of mice heterozygous for different recessive lethal alleles. We find that three different recessive lethal complementation groups are present at the agouti locus, two of which are contained within the al deletion.     

Effects of the lethal yellow (Ay) mutation in mouse aggregation chimeras

The Ay allele is a recessive lethal mutation at the mouse agouti locus, which results in embryonic death around the time of implantation. In the heterozygous state, Ay produces several dominant pleiotropic effects, including an increase in weight gain and body length, a susceptibility to hepatic, pulmonary and mammary tumors, and a suppression of the agouti phenotype, which results in a yellow coat color. To investigate the cellular action of Ay with regard to its effects upon embryonic viability and adult-onset obesity, we generated a series of aggregation chimeras using embryos that differ in their agouti locus genotype. Embryos derived from Ay/a x Ay/a matings were aggregated with those derived from A/A x A/A matings, and genotypic identification of the resultant chimeras was accomplished using a molecular probe at the Emv-15 locus that distinguishes among the three different alleles, Ay, A, and a. Among 50 chimeras, 25 analyzed as liveborns and 25 as 9.5 day embryos, 29 were a/a in equilibrium A/A and 21 were Ay/a in equilibrium A/A. The absence of Ay/Ay in equilibrium A/A chimeras demonstrates that Ay/Ay cells cannot be rescued in a chimeric environment, and the relative deficiency of Ay/a in equilibrium A/A chimeras suggests that, under certain conditions, Ay heterozygosity may partially affect cell viability or proliferation. In the 25 liveborn chimeras, Ay/a in equilibrium A/A animals became obese as adults and a/a in equilibrium A/A animals did not. There was no correlation between genotypic proportions and rate of weight gain, which shows that, with regard to its effects on weight gain, Ay heterozygosity is cell non-autonomous.     

Coupled Site-Directed Mutagenesis/Transgenesis Identifies Important Functional Domains of the Mouse Agouti Protein

The agouti locus encodes a novel paracrine signaling molecule containing a signal sequence, an N-linked glycosylation site, a central lysine-rich basic domain, and a C-terminal tail containing 10 cysteine (Cys) residues capable of forming five disulfide bonds. When overexpressed, agouti causes a number of pleiotropic effects including yellow coat and adult-onset obesity. Numerous studies suggest that agouti causes yellow coat color by antagonizing the binding of α-melanocyte-stimulating hormone (α-MSH) to the α-MSH-(melanocortin-1) receptor. With the goal of identifying functional domains of agouti important for its diverse biological activities, we have generated 14 agouti mutations by in vitro site-directed mutagenesis and analyzed these mutations in transgenic mice for their effects on coat color and obesity. These studies demonstrate that the signal sequence, the N-linked glycosylation site, and the C-terminal Cys residues are important for full biological activity, while at least a portion of the lysine-rich basic domain is dispensable for normal function. They also show that the same functional domains of agouti important in coat color determination are important for inducing obesity, consistent with the hypothesis that agouti induces obesity by antagonizing melanocortin binding to other melanocortin receptors.     

Opposite Orientations of an Inverted Duplication and Allelic Variation at the Mouse Agouti Locus

The mouse agouti protein is a paracrine signaling molecule that causes yellow pigment synthesis. A pale ventral coloration distinguishes the light-bellied agouti (A(w)) from the agouti (A) allele, and is caused by expression of ventral-specific mRNA isoforms with a unique 5' untranslated exon. Molecular cloning demonstrates this ventral-specific exon lies within a 3.1-kb element that is duplicated in the opposite orientation 15-kb upstream to produce an interrupted palindrome and that similarity between the duplicated elements has been maintained by gene conversion. Orientation of the palindrome is reversed in A compared to A(w), which suggests that mutation from one allele to the other is caused by intrachromosomal homologous recombination mediated by sequences within the duplicated elements. Analysis of 15 inbred strains of laboratory and wild-derived mice with Southern hybridization probes and closely linked microsatellite markers suggests six haplotype groups: one typical for most strains that carry A(w) (129/SvJ, LP/J, CE/J, CAST/Ei), one typical for most strains that carry A (Balb/cJ, CBA/J, FVB/N, PERA/Rk, RBB/Dn); and four that are atypical (MOLC/Rk, MOLG/Dn, PERA/Ei, PERC/Ei, SPRET/Ei, RBA/Dn). Our results suggest a model for molecular evolution of the agouti locus in which homologous recombination can produce a reversible switch in allelic identity.     

Recessive yellow in the Mongolian gerbil (Meriones unguiculatus)

A new autosomal recessive coat color mutant in the Mongolian gerbil (Meriones unguiculatus) is described: recessive yellow. On the dorsal side the mutant has a rich yellow to ginger color. Ventrally it shows the typical creamy white belly of a wild-type Mongolian gerbil. The dorsal yellow hairs have short black tips, and a light olive green base. A clear demarcation line between dorsal and ventral color is present. Crosses between recessive yellow animals and multiple homozygous recessive tester animals (a/a; c^chm/c^chm; g/g; p/p) resulted only in animals of an agouti (wild-type) phenotype, showing that the new allele is not allelic with any of the known coat color mutations in the Mongolian gerbil. Molecular studies showed that the new mutant is caused by a missence mutation at the extension (E) locus. On a non-agouti background (a/a; e/e) mutant animals look like a dark wild-type agouti. In contrast to wild-type agouti it shows yellow pigmentation and dark ticking at the ventral side, resulting in the absence of a demarcation line. Since black pigment is present in both the agouti and non-agouti variant (A/A; e/e and a/a; e/e), we conclude that recessive yellow in the Mongolian gerbil is non-epistatic to agouti. Additionally we describe a second mutation at the same locus leading to a similar phenotype, however without black pigment and diminishing yellow pigment during life. Fertility and viability of both new mutants are within normal range. The extension (E) gene is known to encode the melanocortin 1 receptor (MC1R). Interestingly, this is the only gene that is known to account for substantial variation in skin and hair color in humans. Many different mutations are known of which some are associated with higher skin cancer incidence.     

Synteny on mouse chromosome 5 of homologs for human DNA loci linked to the Huntington disease gene

Comparative mapping in man and mouse has revealed frequent conservation of chromosomal segments, offering a potential approach to human disease genes via their murine homologs. Using DNA markers near the Huntington disease gene on the short arm of chromosome 4, we defined a conserved linkage group on mouse chromosome 5. Linkage analyses using recombinant inbred strains, a standard outcross, and an interspecific backcross were used to assign homologs for five human loci, D4S43, D4S62, QDPR, D4S76, and D4S80, to chromosome 5 and to determine their relationships with previously mapped markers for this autosome. The relative order of the conserved loci was preserved in a linkage group that spanned 13% recombination in the interspecific backcross analysis. The most proximal of the conserved markers on the mouse map, D4S43h, showed no recombination with Emv-1, an endogenous ecotropic virus, in 84 outcross progeny and 19 recombinant inbred strains. Hx, a dominant mutation that causes deformities in limb development, maps approximately 2 cM proximal to Emv-1. Since the human D4S43 locus is less than 1 cM proximal to HD near the telomere of chromosome 4, the murine counterpart of the HD gene might lie between Hx and Emv-1 or D4S43h. Cloning of the region between these markers could generate new probes for conserved human sequences in the vicinity of the HD gene or possibly candidates for the murine counterpart of this human disease locus.     

Emv-13 (Akv-3): a noninducible endogenous ecotropic provirus of AKR/J mice

All AKR/J mice carry at least three endogenous ecotropic viral loci which have been designated Emv-11 (Akv-1), Emv-13 (Akv-3), and Emv-14 (Akv-4) (Jenkins et al., J. Virol. 43:26-36, 1982.) Using two independent AKR/J-derived sets of recombinant inbred mouse strains, AKXL (AKR/J x C57L/J) and AKXD (AKR/J x DBA/2J), as well as the HP/EiTy strain (an Emv-13-carrying inbred strain partially related to AKR/J mice) (Taylor et al., J. Virol. 23:106-109, 1977), we have examined the association of these endogenous viral loci with virus expression. Strains which transmit Emv-11 or Emv-14 or both were found to produce virus spontaneously, whereas strains that transmit Emv-13 alone were negative for virus expression. Restriction endonuclease digestion and hybridization with an ecotropic virus-specific hybridization probe of DNAs from strains which transmit only Emv-13 yielded enzyme cleavage patterns identical to those observed with DNAs from strains transmitting Emv-11 or Emv-14 or both. These findings indicate the absence of any gross rearrangement of Emv-13 proviral sequences. Cell cultures derived from recombinant inbred strains that carry only Emv-13 failed to express detectable infectious virus, viral proteins, or cytoplasmic ecotropic virus-specific RNA even after treatment with 5-iodo-2-deoxyuridine or 5-azacytidine, an inhibitor of DNA methylation. Our results indicate that a mechanism(s) other than methylation of Emv-13 proviral DNA is responsible for inhibition of Emv-13 expression.     

Genetic determinants of sable and umbrous coat color phenotypes in mice

The dorsal fur in yellow F1 mice (F1-Ay) between C3H/HeJ and C57BL/6J-Ay is darker than that in C57BL/6J-Ay. Moreover, yellow F2 mice (F2-Ay) exhibit a wide spectrum of coat color phenotypes in terms of lightness and darkness. Quantitative trait locus (QTL) analysis on F2-Ay identified three significant modifier loci that accounted for darkening of the coat color on chromosomes 1 (Dmyaq1 and Dmyaq2) and 15 (Dmyaq3), and the C3H/HeJ allele at these loci increased the darkness. Because agouti F2 mice (F2-A) also exhibited a spectrum of coat color phenotypes, the question of whether these QTLs had any effects on F2-A was examined. Dmyaq1 and Dmyaq2 were shown to increase the darkness in F2-A, whereas Dmyaq3 did not. The results showed that Dmyaq1-Dmyaq3 were parts of determinants responsible for the sable (darker modification of yellow) coat color phenotype, and that Dmyaq1 and Dmyaq2 were parts of determinants responsible for the umbrous (darker modification of agouti) coat color phenotype. It is, thus, demonstrated that both the sable and the umbrous phenotypes resulted from multigenic contributions, and that they shared genetic bases, as had been implied for several decades.     

Chromosomal localization of Emv-16 and Emv-17, two closely linked ecotropic proviruses of RF/J mice.

Emv-16 and Emv-17, the two closely linked ecotropic proviral loci of RF/J mice, have been mapped to chromosome 1 between leaden, ln, and the mouse engrailed homeo-box locus, En-1, by using recombinant inbred strains and conventional backcross analysis.     

Circling,deafness, and yellow coat displayed by yellow submarine (ysb) and light coat and circling (lcc) mice with mutations on chromosome 3

We describe here two mouse mutants, yellow submarine (Ysb) and light coat and circling (Lcc). Ysb arose as the result of insertions of a transgene, pAA2, into the genome. Lcc is an independent, radiation-induced mutation. Both mutants are characterized by recessive circling behavior and deafness, associated with a non-segregating, semi-dominant yellow coat color. Complementation tests showed that Ysb and Lcc are allelic. We attribute the yellow coat in Ysb and Lcc mice to the absence of black awl overhairs, increased agouti zigzag underhairs, and the presence of agouti awls with long subapical yellow pigment. Chromosomal mapping and genomic characterization showed the Ysb and Lcc mutations involve complex chromosomal rearrangements in overlapping regions of mouse chromosome 3, A2/A3-B/C and B-E1, respectively. Ysb and Lcc show for the first time, to our knowledge, the presence of genes in the B-C region of chromosome 3 important for balance and hearing and the pigmentation and specification of coat hair.     

Regulation of yellow pigment formation in mice: a historical perspective

Pigment synthesis by hair follicle melanocytes is modulated by a large number of environmental and genetic factors, many of which are discussed in this review. Eumelanic (non-yellow) pigment is produced by hair follicle melanocytes following the binding of alpha-melanocyte stimulating hormone to melanocortin receptor 1. Binding of this hormone to the melanocyte membrane is blocked by agouti signaling protein (ASP) which is encoded by the agouti locus and results in the synthesis of yellow pigment, instead of non-yellow (black/brown) pigment. The cyclical release of ASP by hair follicle cells results in a black/brown hair with a subapical yellow band. This is the wild-type coat color pattern of many mammals and is called agouti. Several dominant mutations at the agouti locus in mice, induced by retrotransposon-like intracisternal A particles, result in ectopic over-expression of ASP and animals with much higher proportions of all-yellow hairs. This abnormal presence of ASP in essentially all body cells results in the 'yellow agouti obese mouse syndrome.' The obesity has been associated with binding of ASP to melanocortin receptor 4 inactivating the latter. The syndrome also includes hyperinsulinemia, increased somatic growth, and increased susceptibility to hyperplasia and carcinogenesis. The physiologic and molecular bases for these syndrome components have not yet been elucidated. This historically orientated review is subdivided, where applicable, into pre- and post-1992 subsections to emphasize the impact of the cloning of the agouti and extension loci and their protein products on the identification of the molecular and physiological pathways modulating the manifold aspects of pheomelanogenesis.