Doreen Chambers

A second tyrosinase-related protein, TRP-2 maps to and is mutated at the mouse slaty locus

We have cloned and sequenced mouse cDNAs corresponding to a third member of a family of melanocyte‐specific mRNAs, which encode tyrosinase and related proteins. This new member, tyrosinase‐related protein‐2 (TRP‐2), has approximately 40% amino acid identity with the two other proteins in the family and has the same structural features including two copper binding sites, two cysteine‐rich regions, a signal peptide and a transmembrane domain. We now show that one of the cysteine‐rich regions in this protein family is an ‘EGF‐like’ repeat found in many extracellular and cell surface proteins. The gene encoding TRP‐2 maps to mouse chromosome 14, in the region of the coat colour mutation slaty. We show that the TRP‐2 of slaty mice has a single amino acid difference from wild‐type TRP‐2; a substitution of glutamine for arginine in the first copper binding site. TRP‐2 is the much sought melanogenic enzyme DOPAchrome tautomerase (DT), which catalyses the conversion of DOPAchrome to 5,6,dihydroxyindole‐2‐carboxylic acid. Extracts from mice homozygous for the slaty mutation have a 3‐fold or more reduction in DT activity, indicating that TRP‐2/DT is encoded at the slaty locus, and the missense mutation reduces but does not abolish the enzyme activity.

Mutations in Sox18 underlie cardiovascular and hair follicle defects in ragged mice.

Analysis of classical mouse mutations has been useful in the identification and study of many genes. We previously mapped Sox18, encoding an SRY-related transcription factor, to distal mouse chromosome 2 (ref. 2). This region contains a known mouse mutation, ragged (Ra), that affects the coat and vasculature. Here we have directly evaluated Sox18 as a candidate for Ra. We found that Sox18 is expressed in the developing vascular endothelium and hair follicles in mouse embryos. Furthermore, we found no recombination between Sox18 and Ra in an interspecific backcross segregating for the Ra phenotype. We found point mutations in Sox18 in two different Ra alleles that result in missense translation and premature truncation of the encoded protein. Fusion proteins containing these mutations lack the ability to activate transcription relative to wild-type controls in an in vitro assay. Our observations implicate mutations in Sox18 as the underlying cause of the Ra phenotype, and identify Sox18 as a critical gene for cardiovascular and hair follicle formation.

Genomic structure and chromosomal mapping of the murine and human Mbd1, Mbd2, Mbd3, and Mbd4 genes

Abstract. DNA methylation is essential for murine development and is implicated in the control of gene expression. MeCP2, MBD1, MBD2, MBD3, and MBD4 comprise a family of mammalian, nuclear proteins related by the presence in each of an amino acid motif called the methyl-CpG binding domain (MBD). Each of these proteins, with the exception of MBD3, is capable of binding specifically to methylated DNA. MeCP2, MBD1 and MBD2 can also repress transcription. We describe the genomic structure and chromosomal localization of the human and murine Mbd1, Mbd2, Mbd3, and Mbd4 genes. We find that the highly similar MBD2 and MBD3 proteins are encoded by genes that map to different chromosomes in humans and mice but show a similar genomic structure. The Mbd1 and Mbd2 genes, in contrast, map together to murine and human Chromosomes (Chrs)18. The Mbd3 and Mbd4 genes map to murine Chrs 10 and 6, respectively, while the human MBD3 and MBD4 genes map to Chrs 19 and 3, respectively.

Analysis of CAG trinucleotide repeats from mouse cDNA sequences

A number of human single gene disorders are now known to result from abnormal expansion of trinucleotide repeats. Spinal muscular bulbar atrophy, myotonic dystrophy, Huntingtons Disease, spinocerebellar ataxia and dentatorubral‐pallidoluysian atrophy are all caused by expansions of CAG repeats. Abnormal expansion of trinucleotide repeats has only so far been described in humans, and no mouse models exist for these diseases. In order to investigate trinucleotide repeat stability in mice, the Genbank and EMBL nucleotide databases were screened to find genes containing CAG repeats. Of the sequences selected, 32 were from mouse, and in 12 of these the repeat was in transcribed sequence and contained at least seven perfect repeats. These repeats were then analysed by PCR to evaluate the degree of variability of repeat length in the various genes. Two of the genes containing variable length CAG repeats, seven in absentia homologue 1b (Sinh1b), and choline acetyl transferase (Chat), which had not previously been mapped in the mouse genome, were mapped by linkage analysis in an interspecific backcross. Sinh1b maps very distally on the X chromosome, and Chat maps to chromosome 14.