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.     

Molecular Characterization of the pun Allele of the Mouse Pink-Eyed Dilution Locus

The mouse pink eyed dilution locus, p, located on chromosome 7, mediates coat and eye color. The human correlate of this gene may underlie some forms of tyrosinase-positive oculocutaneous albinism. Mutations at the p locus result in a reduction in pigmentation of the eyes and coat. Although most mutant p alleles (including all spontaneous mutations) affect only pigmentation, several mutant alleles (all radiation induced) are also associated with a variety of other phenotypes. We have focused our attention on the p^un mutant allele, a spontaneous mutation, exhibiting one of the highest reversion frequencies reported for a mammalian mutation. Using a new technique, genome scanning, we have cloned fragments of genomic DNA from the p locus that are associated with a DNA duplication in p^un DNA. These fragments can now be used to locate the p gene-encoding sequences and aid in the molecular characterization of complex mutant p alleles.     

Influence of Maternal Phenotype on Metabolic Differentiation of Agouti Locus Mutants in the Mouse

The results of extensive breeding experiments indicate that the phenotypic differentiation of embryos carrying the viable yellow, A ^vy, or mottled, a^m, mutations is influenced to a major extent by the agouti locus genotype and the strain genome of the dam. The A^vy/a and a^m/a genotypes are each expressed in a spectrum of coat color phenotypes. These can be grouped into two classes, mottled and pseudoagouti.—In a reciprocal cross of C57BL/6JNIcrWf and AM/Wf-a^m/a^m mice, 29.5% of the offspring of C57BL/6 dams were of the pseudoagouti phenotype, whereas no pseudoagouti offspring were produced by AM strain dams.—Mottled yellow A^vy/a mice become obese and tumor formation is enhanced in these mice in comparison with the lean pseudoagouti A^vy/a siblings.—In two different reciprocal crosses using four different inbred strains, the proportion of pseudoagouti A^vy/a offspring differed according to the strain of the dam. Regardless of strain, mottled yellow A ^vy/a dams produced significantly fewer pseudoagouti A ^vy/a offspring than did black a/a dams.—The data suggest that metabolic differentiation of A^vy/a zygotes into phenotypic classes with different susceptibilities to obesity and tumor formation is influenced to a considerable degree by the metabolic characteristics of the oviductal and uterine environment of the dam.     

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.     

Genetic and Physical Mapping of the Mouse Ulnaless Locus

The mouse Ulnaless locus is a semidominant mutation which displays defects in patterning along the proximal-distal and anterior-posterior axes of all four limbs. The first Ulnaless homozygotes have been generated, and they display a similar, though slightly more severe, limb phenotype than the heterozygotes. To create a refined genetic map of the Ulnaless region using molecular markers, four backcrosses segregating Ulnaless were established. A 0.4-cM interval containing the Ulnaless locus has been defined on mouse chromosome 2, which has identified Ulnaless as a possible allele of a Hoxd cluster gene(s). With this genetic map as a framework, a physical map of the Ulnaless region has been completed. Yeast artificial chromosomes covering this region have been isolated and ordered into a 2 Mb contig. Therefore, the region that must contain the Ulnaless locus has been defined and cloned, which will be invaluable for the identification of the molecular nature of the Ulnaless mutation.     

Characterization of Mutations at the Mouse Phenylalanine Hydroxylase Locus

Two genetic mouse models for human phenylketonuria have been characterized by DNA sequence analysis. For each, a distinct mutation was identified within the protein coding sequence of the phenylalanine hydroxylase gene. This establishes that the mutated locus is the same as that causing human phenylketonuria and allows a comparison between these mouse phenylketonuria models and the human disease. A genotype/phenotype relationship that is strikingly similar to the human disease emerges, underscoring the similarity of phenylketonuria in mouse and man. InPAH^ENU1,the phenotype is mild. ThePah^enu1mutation predicts a conservative valine to alanine amino acid substitution and is located in exon 3, a gene region where serious mutations are rare in humans. InPAH^ENU2,the phenotype is severe. ThePah^enu2mutation predicts a radical phenylalanine to serine substitution and is located in exon 7, a gene region where serious mutations are common in humans. InPAH^ENU2,the sequence information was used to devise a direct genotyping system based on the creation of a newAlw26I restriction endonuclease site.     

Recessive Alleles Found at R and C Loci in Maize Stocks Showing Aberrant Ratio at the A Locus

Corn stocks showing virus-induced aberrant ratio (AR) at the "A" locus were found to have recessive alleles at the R and/or C loci. Since by the known pedigree these loci should be homozygous dominant, the results suggest an inactivation of maize genes by a mechanism as yet unknown. The presence of recessive alleles at these additional loci can explain the segregation ratios obtained in these particular stocks.     

Molecular Markers for the agouti Coat Color Locus of the Mouse

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-15 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 H1 of chromosome 2, thus directly confirming previous assignments of the location of the agouti locus.     

Characterization of Mouse Pmel 17 Gene and Silver Locus

Pmel 17 cDNA clones, isolated from wild-type and si/si murine melanocytes, were sequenced and compared. A single nucleotide (A) insertion was found in the putative cytoplasmic tail of the si/si Pmel 17 cDNA clone. This insertion is predicted to alter the last 24 amino acids at the C-terminus and to extend the Pmel 17 protein by 12 residues. The mutation was confirmed by the sequence of the PCR-amplified genomic region including the mutation site. Silver Pmel 17 was not recognized by antibodies directed toward the C-terminal amino acids of wild-type Pmel 17, indicating a defect in this region. These results indicate that silver Pmel 17 protein has a major defect at the carboxyl terminus.     

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.     

A High-Resolution Genetic Map of the Nervous Locus on Mouse Chromosome 8

The nervous (nr) mutant mouse displays two gross recessive traits: both an exaggeration of juvenile hyperactivity and a pronounced ataxia become apparent during the third and fourth postnatal weeks. Using an intersubspecific intercross, we have established a high-resolution map of a segment of mouse Chromosome 8 that places thenrlocus in a genomic segment defined byD8Rck1on the centromeric end andD8Mit3on the telomeric end. This map position places thenrlocus within the BALB/cGr congenic region of the C3HeB/FeJ-nrstrain, confirming the accuracy of our study. We used this map position to identify and evaluate three genes—ankyrin 1, cortexin, and farnesyltransferase—as candidates for thenrgene. These three genes were eliminated from consideration but allowed us to establish the conservation of synteny between the region containing thenrlocus and a segment of the short arm of human chromosome 8 (8p21–p11.2). Finally, the incomplete penetrance of thenrphenotype led us to perform a screen for modifier loci, and we present evidence that such a nervous modifier locus may exist on mouse Chromosome 5.     

Genetic and Physical Mapping of the Dreher Locus on Mouse Chromosome 1

Mutations in the mouse dreher (dr) gene cause skeletal defects, hyperactivity, abnormal gait, deafness, white belly spotting, and hypoplasia of Müllerian duct derivatives. To map dr to high resolution, we utilized two crosses. Initially, we analyzed an intersubspecific intercross to construct a detailed genetic map of simple sequence length polymorphism markers within a 6.3-cM region surrounding the dr locus. Subsequently, we analyzed a second intersubspecific intercross segregating for the dr(6J) allele, which positioned dr within a 0.13-cM region between Rxrg and D1Mit370. A physical contig of BAC clones spanning the dr critical region was constructed, and eight potential dr candidate genes were excluded by genetic or physical mapping. Together these results lay the foundation for positional cloning of the dr gene.