Joseph H. Nadeau

Pigmentation phenotypes of variant extension locus alleles result from point mutations that alter MSH receptor function

Coat colors in the chestnut horse, the yellow Labrador retriever, the red fox, and one type of yellow mouse are due to recessive alleles at the extension locus. Similarly, dominant alleles at this locus are often responsible for dark coat colors in mammals, such as the melanic form of the leopard, Panthera pardus. We show here that the murine extension locus encodes the melanocyte-stimulating hormone (MSH) receptor. In mice, the recessive yellow allele (e) results from a frameshift that produces a prematurely terminated, nonfunctioning receptor. The sombre (Eso and Eso-3J) and tobacco darkening (Etob) alleles, which both have dominant melanizing effects, results from point mutations that produce hyperactive MSH receptors. The Eso-3J receptor is constitutively activated, while the Etob receptor remains hormone responsive and produces a greater activation of its effector, adenylyl cyclase, than does the wild-type allele.

Mapping of the ACTH, MSH, and neural (MC3 and MC4) melanocortin receptors in the mouse and human

The melanocortin peptides regulate a wide variety of physiological processes, including pigmentation and glucocorticoid production, and also have several activities in the central and peripheral nervous systems. The melanocortin receptor family includes the melanocytestimulating hormone receptor (MSH-R), adrenocorticotropic hormone receptor (ACTH-R), and two neural receptors, MC3-R and MC4-R. In the human these receptors map to 16q24 (MSH-R), 18p11.2 (ACTH-R), 20q13.2 (MC3-R), and 18q22 (MC4-R). The corresponding locations in the mouse are 8, 18, and 2; a variant for mapping MC4-R has not yet been identified. The data reported here also show that the neural MC3 receptor maps close to a disease locus for benign neonatal epilepsy in human and near the El-2 epilepsy susceptibility locus in the mouse.

Multilocus markers for mouse genome analysis: PCR amplification based on single primers of arbitrary nucleotide sequence

Polymerase chain reaction (PCR) based on single primers of arbitrary nucleotide sequence provides a powerful marker system for genome analysis because each primer amplifies multiple products, and cloning, sequencing, and hybridization are not required. We have evaluated this typing system for the mouse by identifying optimal PCR conditions; characterizing effects of GC content, primer length, and multiplexed primers; demonstrating considerable variation among a panel of inbred strains; and establishing linkage for several products. Mg2+, primer, template, and annealing conditions were identified that optimized the number and resolution of amplified products. Primers with 40% GC content failed to amplify products readily, primers with 50% GC content resulted in reasonable amplification, and primers with 60% GC content gave the largest number of well-resolved products. Longer primers did not necessarily amplify more products than shorter primers of the same proportional GC content. Multiplexed primers yielded more products than either primer alone and usually revealed novel variants. A strain survey showed that most strains could be readily distinguished with a modest number of primers. Finally, linkage for seven products was established on five chromosomes. These characteristics establish single primer PCR as a powerful method for mouse genome analysis.

3-Hydroxy-3-methylglutaryl coenzyme A lyase (HL): cloning and characterization of a mouse liver HL cDNA and subchromosomal mapping of the human and mouse HL genes

Abstract3-Hydroxy-3-methylglutaryl coenzyme A lyase (HL) is a homodimeric mitochondrial matrix enzyme that catalyzes the last step of ketogenesis. Using a human HL cDNA as a probe, we isolated a 1.4-kb mouse HL cDNA (HLM) from a mouse liver library and extended the sequence in the 5′ direction, using RACE PCR to include the complete coding sequence. The nucleotide sequence of the mouse HL coding region is 85.7% identical to human HL, and 52.6% to Ps. mevalonii HL. Peptide identities of 87.4% and 54.3% respectively were observed. Southern analysis of 29 strains of laboratory mice and of Mus spretus revealed a total of about 25 kb of hybridizing fragments and three polymorphic fragments in both EcoRI and HindIII digestions. The mouse HL locus (Hmgcl) was localized on Chromosome (Chr) 4: Pmv-19-12.6±3.6 cM-Hmgcl-7.3±2.3 cM-Xmv-8-1.5±1.0 cM-Gpd1. The human HL locus (HMGCL) was mapped to distal Chr 1p by analysis of a human-hamster hybrid cell panel and by in situ hybridization.

Close linkage of retinoic acid receptor genes with homeobox- and keratin-encoding genes on paralogous segments of mouse chromosomes 11 and 15.

Retinoic acid is essential for normal development and growth of structures such as head and limbs, and it can act as morphogen or teratogen. Retinoic acid induces expression of genes such as the homeobox genes and keratin type I and type II genes. Retinoic acid receptors are nuclear transcription factors that play a key role in retinoid physiology. As part of the characterization of retinoic acid receptor gene family, linkage of genes encoding the three receptors was determined by using interspecific backcross and recombinant inbred strain analysis of restriction fragment variants. Retinoic acid receptor α is located on mouse Chromosome (Chr) 11 near the homeobox-2 complex and the keratin type I gene complex, whereas retinoic acid receptor γ is on mouse Chr 15 near the homeobox-3 complex and the keratin type II complex. Close genetic proximity of these functionally related genes may be significant. We confirmed assignment of retinoic acid receptor β to the centromeric portion of Chr 14. These linkage assignments provide further evidence for duplicated segments in the mouse genome.

New murine polymorphisms detected by random amplified polymorphic DNA (RAPD) PCR and mapped by use of recombinant inbred strains

Oligonucleotide primers of random sequence that were 12 bases in length, 58% in GC content, and lacking internal palindromes were designed. By random amplified polymorphic DNA (RAPD) PCR, these primers were used to survey for DNA variations between the progenitors of the mouse AXB and BXA recombinant inbred sets (A/J and C57BL/6J). We identified 17 DNA variants detected by 10 primers. Map positions for these variants were determined by comparing their strain distribution patterns in the AXB, BXA recombinant inbred sets with strain distribution patterns of previously published loci. When necessary, BXD and NXSM recombinant inbred sets were also used. These 17 new loci mapped to 12 chromosomes. The 10 primers were also used to survey 20 inbred mouse strains including the progenitors of other recombinant inbred sets and four mouse strains recently inbred from the wild (CAST/Ei, MOLF/Ei, PERA/Ei, and SPRET/Ei).

Mouse map of paralogous genes

Table 1. Paralogous genes. marked by members of the myos in heavy chain gene family and the acetylcholine receptor gene family. The skeletal form of the myosin gene (Myhs) and the gene encoding the [3 chain of the acetylcholine recep tor (Acrb) are closely linked on Chromosome (Chr) 11. Similarly, the cardiac form of the myosin heavy chain gene (Myhc) and one of the genes encoding the et chain of the ace ty lchol ine r ecep to r (Acra-2) are c lose ly linked on Chr 14. Inspect ion of the genome map of paralogous genes (Fig. 1) reveals other examples of duplicated segments.

Mapping of the murine and rat Facc genes and assessment of flexed-tail as a candidate mouse homolog of Fanconi anemia group C.

Fanconi anemia is a rare, autosomal recessive disorder characterized at the cellular level by a combination of hypersensitivity to DNA-damaging agents and chromosomal instability. Clinical features include pancytopenia, often associated with specific congenital malformations, and a predisposition to leukemia. We previously cloned the gene defective in Fanconi anemia group C by complementation of the intrinsic sensitivity of Fanconi anemia cells to DNA cross-linking agents, and we recently cloned its mouse homolog (Facc). In this report, we localized Facc to mouse Chromosome (Chr) 13 and its rat homolog to rat Chr 17. A previously described anemic mouse mutant, flexed-tail, maps to the same chromosomal region. Differences were detected between DNA of the flexed-tail and congenic mice, indicating the proximity of the Facc probe to the disease mutation. Analysis of flexed-tail RNA did not reveal detectable difference in Facc message level or size between flexed-tail and congenic mice. On this basis, we conclude that, although flexed-tail remains a candidate for Fanconi anemia in the mouse, there is no evidence currently that Facc is mutated in flexed-tail mice.

The mouse genome: an overview

A genetic map with one molecularly marked locus per cM will be available for the mouse in the near future. A map of this density should provide molecular reference points that connect genetic and physical maps, identify sites to initiate positional cloning studies for the molecular characterization of mutant loci, and define homologous regions of mouse and human genomes.

Meeting Report: Fourth International Workshop on Mouse Genome Mapping

This was the fourth in a series of meetings which have taken place annually within the international community of scientists who have a special interest in mapping the mouse genome. Previous workshops had brought the community together to review the status of the known linkages and the current progress in the development of genetic maps. Those efforts were very helpful and at the close of the Third Workshop held in Oxford, UK, August 1989, there was a broad interest in formalizing committees from the research community to review the existing genome information. The initial efforts to establish chromosome-specific committees was accomplished before the 1990 workshop and the work of the committees was successfully initiated in Annapolis. Reports on the current status of the map of each chromosome (Chr) are presently being prepared for publication in a separate issue of Mammalian Genome in 1991. In addition, the workshop was able to articulate a set of common research goals for mapping the mouse genome which reflect the unique strengths and value of the mouse as an experimental system for genetic studies. These goals include saturated genetic maps based on well-spaced reference loci, physical maps of selected chromosome segments, and the development and dissemination of mouse genomic databases. Progress can be assessed by comparing the consensus statement of research goals that emerged from the meeting with the results that were actually presented.