Hundreds of murine dilute mutations have been identified and analysed, making dilute one of the best genetically characterized of all mammalian loci. The recessive dilute (d) coat colour mutation carried by many inbred strains of mice produces a lightening of coat colour, caused by an abnormal adendritic melanocyte morphology that results in an uneven release of pigment granules into the developing hair shaft. Most dilute alleles (dilute-lethal) also produce a neurological defect, characterized by convulsions and opisthotonus, apparent at 8-10 days of age and continuing until the death of the animal at 2-3 weeks of age. The discovery that the original dilute allele (now termed dilute-viral or dV) is the result of the integration of an ecotropic murine leukaemia provirus has allowed the cloning of genomic DNA and in this study complementary DNA, from the dilute locus. The predicted dilute amino-acid sequence indicates that dilute encodes a novel type of myosin heavy chain, with a tail, or C-terminal, region that has elements of both type II (alpha-helical coiled-coil) and type I (non-coiled-coil) myosin heavy chains. Dilute transcripts are differentially expressed in both embryonic and adult tissues and are very abundant in neurons of the central nervous system, cephalic ganglia, and spinal ganglia. These results suggest an important role for the dilute gene product in the elaboration, maintenance, or function of cellular processes of melanocytes and neurons.

The murine dilute coat color locus encodes an unconventional myosin heavy chain that is thought to be required for the elaboration or maintenance of dendrites or organelle transport in melanocytes and neurons. In previous studies we showed that the d mutation carried by many inbred strains of mice (now referred to as dilute viral, dr), is caused by the integration of an ecotropic murine leukemia virus (Emv-3) into the dilute gene and that phenotypic revertants of dv (termed d+) result from viral excision; a solo viral long terminal repeat (LTR) is all that remains in revertant DNA. In the studies described here we show that Emv-3 sequences are located within an intron of the dilute gene in a region of the Cterminal tail that is differentially spliced. We also show that these Emv-3 sequences result in the production of shortened and abnormally spliced dilute transcripts and that the level of this effect varies among tissues. This tissue-specific effect on dilute expression likely accounts for the absence of neurological abnormalities observed in dV mice. Surprisingly, we also found that the solo viral LTR present in revertant d+ DNA produces a tissue-specific effect on dilute expression, although this effect is less dramatic than with the full-length provirus and produces no obvious mutant phenotype. These findings have important implications for understanding the effects of viral sequences on mammalian gene expression.

The Saccharomyces cerevisiae leucine-inserting amber suppressor tRNA gene SUP53 (a tRNA Le allele) was used to investigate the relationship between precursor tRNA structure and mature tRNA function. This gene encodes a pre-tRNA which contains a 32-base intron. The mature tRNASuPS3 contains a 5-methylcytosine modification of the anticodon wobble base. Mutations were made in the SUP53 intron. These mutant genes were transcribed in an S. cerevisiae nuclear extract preparation. In this extract, primary tRNA gene transcripts are end-processed and base modified after addition of cofactors. The base modifications made in vitro were examined, and the mutant pre-tRNAs were analyzed for their ability to serve as substrates for partially purified S. cerevisiae tRNA endonuclease and ligase. Finally, the suppressor function of these mutant tRNA genes was assayed after their integration into the S. cerevisiae genome. Mutant analysis showed that the totally intact precursor tRNA, rather than any specific sequence or structure of the intron, was necessary for efficient nonsense suppression by tRNASuPS3. Less efficient suppressor activity correlated with the absence of the 5-methylcytosine modification. Most of the intron-altered precursor tRNAs were successfully spliced in vitro, indicating that modifications are not critical for recognition by the tRNA endonuclease and ligase.Intervening sequences (IVS), or introns, have been found in the genes which encode the major classes of eucaryotic RNA, both nuclear and organellar. Recently, the mechanisms by which introns are removed from the primary RNA transcript have been the focus of intense investigation (reviewed in references 7, 14, and 51). However, relatively few experiments have elucidated a biological function for an IVS separate from the role of intron sequence or structure in the splicing process.Saccharomyces cerevisiae affords an ideal system for investigating intron function. A number of yeast nuclear mRNA and tRNA genes containing introns have been cloned. These genes can be mutated in vitro and the mutant phenotype assayed by introducing the mutated genes into yeast cells by chromosomal integration. Using these methods, Ng et al. (40) showed that precise removal of the S. cerevisiae actin gene intron did not affect the production of actin mRNA or actin protein in vivo. Cells containing only the intron-deleted actin gene exhibit a normal growth rate and normal meiotic products upon sporulation.In contrast, Johnson and Abelson (22) found that precise removal of the intron from an S. cerevisiae suppressor tRNA gene has profound effects on the phenotype of the mature suppressor tRNA product. Comparing strains containing the tyrosine-inserting ocher suppressor tRNASUP6 and its introndeleted counterpart SUP6A32, they found that SUP6A32 was inefficient in suppressing ocher nonsense mutations.When in vivo-labeled tRNATYr from the two strains was examined, SUP6-specific RNA from the deletion strain its total structure or some specific feature of the IVS itself, is recognized a...

Three mutations of the enzyme dihydrofolate reductase were constructed by oligonucleotide-directed mutagenesis of the cloned Escherichia coli gene. The mutations--at residue 27, aspartic acid replaced with asparagine; at residue 39, proline replaced with cysteine; and at residue 95, glycine replaced with alanine--were designed to answer questions about the relations between molecular structure and function that were raised by the x-ray crystal structures. Properties of the mutant proteins show that Asp-27 is important for catalysis and that perturbation of the local structure at a conserved cis peptide bond following Gly-95 abolishes activity. Substitution of cysteine for proline at residue 39 results in the appearance of new forms of the enzyme that correspond to various oxidation states of the cysteine. One of these forms probably represents a species cross-linked by an intrachain disulfide bridge between the cysteine at position 85 and the new cysteine at position 39.

Multi-parent populations (MPPs) capture and maintain the genetic diversity from multiple inbred founder strains to provide a resource for high-resolution genetic mapping through the accumulation of recombination events over many generations. Breeding designs that maintain a large effective population size with randomized assignment of breeders at each generation can minimize the impact of selection, inbreeding, and genetic drift on allele frequencies. Small deviations from expected allele frequencies will have little effect on the power and precision of genetic analysis, but a major distortion could result in reduced power and loss of important functional alleles. We detected strong transmission ratio distortion in the Diversity Outbred (DO) mouse population on chromosome 2, caused by meiotic drive favoring transmission of the WSB/EiJ allele at the R2d2 locus. The distorted region harbors thousands of polymorphisms derived from the seven non-WSB founder strains and many of these would be lost if the sweep was allowed to continue. To ensure the utility of the DO population to study genetic variation on chromosome 2, we performed an artificial selection against WSB/EiJ alleles at the R2d2 locus. Here, we report that we have purged the WSB/EiJ allele from the drive locus while preserving WSB/EiJ alleles in the flanking regions. We observed minimal disruption to allele frequencies across the rest of the autosomal genome. However, there was a shift in haplotype frequencies of the mitochondrial genome and an increase in the rate of an unusual sex chromosome aneuploidy. The DO population has been restored to genome-wide utility for genetic analysis, but our experience underscores that vigilant monitoring of similar genetic resource populations is needed to ensure their long-term utility.

This review compares two novel polygenic mouse models of type 2 diabetes (T2D), TALLYHO/JngJ and NONcNZO10/LtJ, and contrasts both with the well-known C57BLKS/J-Leprdb (db/db) monogenic diabesity model. We posit that the new polygenic models are more representative of the “garden variety” obesity underlying human T2D in terms of their polygenetic rather than monogenic etiology. Moreover, the clinical phenotypes in these new models are less extreme, for example, more moderated development of obesity coupled with less extreme endocrine disturbances. The more progressive development of obesity produces a maturity-onset development of hyperglycemia in contrast to the juvenile-onset diabetes observed in the morbidly obese db/db model. Unlike the leptin receptor-deficient db/db models with central leptin resistance, the new models develop a progressive peripheral leptin resistance and are able to maintain reproductive function. Although the T2D pathophysiology in both TALLYHO/JngJ and NONcNZO10/LtJ is remarkably similar, their genetic etiologies are clearly different, underscoring the genetic heterogeneity underlying T2D in humans.

The dilute (d) coat color locus of mouse chromosome 9 has been identified by more than 200 spontaneous and mutagen-induced recessive mutations. With the advent of molecular probes for this locus, the molecular lesion associated with different dilute alleles can be recognized and precisely defined. In this study, two dilute mutations, dilute-lethal20' (d'a01) and dilute prenatal lethal Aa2, have been examined. Using a dilute locus genomic probe in Southern blot analysis, we detected unique restriction fragments in de0J and Aa2 DNA.Subsequent analysis of these fragments showed that they represented deletion breakpoint fusion fragments. DNA sequence analysis of each mutation-associated deletion breakpoint fusion fragment suggests that both genomic deletions were generated by nonhomologous recombination events. The spontaneous dL'OJ mutation is caused by an interstitial deletion that removes a single coding exon of the dilute gene. The correlation between this discrete deletion and the expression of all dilute-associated phenotypes in F0I homozygotes defines the d"P0 mutation as a functional null allele of the dilute gene. The radiation-induced Aa2 allele is a multilocus deletion that, by complementation analysis, affects both the dilute locus and the proximal prenatal lethal-3 (p1-3) functional unit. Molecular analysis of the Aa2 deletion breakpoint fusion fragment has provided access to a previously undefined gene proximal to d. Initial characterization of this new gene suggests that it may represent the genetically defined pl-3 functional unit.Classic genetic analysis of variant mouse phenotypes has defined numerous genetic loci and, in some cases, numerous alleles at a given locus. However, the physical basis for these mutations and an understanding of the impact of mutagenic events on gene expression have been impeded by difficulties in gaining molecular access to the mutated genes. The murine dilute (d) locus is among those rare loci for which extensive analysis at both the genetic and molecular levels is possible. A vast collection of spontaneous, chemical-induced, and radiation-induced dilute locus mutations have been generated. With the ever-increasing number of genomic and cDNA probes for the dilute locus, the physical detection and precise analysis of DNA alterations and rearrangements associated with individual d alleles can now be accomplished. As illustrated in this study, this combined molecular and genetic analysis is elucidating the gene product(s) and genomic organization not only of the d locus but also of other loci in the d region of mouse chromosome 9.More than 200 spontaneous and induced dilute mutations have been identified (25 duced dilute-opisthotonic (d0P) class exhibit the neurological disorder of opisthotonus, a convulsive arching of the animal's head and neck (30,33,34). These seizures are readily apparent at 9 days postpartum and continue until death at approximately 3 weeks of age. In extensive complementation analysis, the coat color, opisthotonic, and lethality phenotypes are geneti...

At present, the dilute (dV) coat-color mutation of DBA mice provides the only simple means for measuring the relative somatic and germ-line reverse mutation rates of retrovirus-induced mutations in mammals. The d' mutation was generated by the spontaneous integration of an ecotropic murine leukemia virus into noncoding sequences of the dilute locus. Reversion of the d' mutation occurs by provirus excision and is mediated by homologous recombination events involving the viral long terminal repeat sequences. Although numerous independent germ-line d+ revertants have been identified, somatic d+ revertants have not been reported previously. During the past 5 years, we have screened more than one million mice homozygous for the dV mutation to determine whether we could identify somatic d+ revertants. This survey has resulted in the identification of a somatic d+ revertant and has provided a data base from which we can estimate the relative somatic and germ-line excision frequencies of retroviruses in mice and speculate about the nature of homologous recombination events producing d' revertant alleles.The recessive coat-color determinant dilute (d) is an old mutation of the mouse fancy and was among the variants first studied by mouse geneticists in the early 1900s. This original d mutation, which maps to mouse chromosome 9, has been incorporated into several inbred strains, including DBA. The d allele carried by DBA mice has a single phenotypic effect, a lightening of coat color. Dilution of pigment in these mice is the result of an alteration in melanocyte morphology; the amount of melanin synthesized is normal (1). In large breeding populations of DBA mice, it is possible to identify reverse mutations (reversions) of the d mutation to wild-type phenotype (d+). These reversions are germinal and dominant. They are identified as intensely colored mice in an otherwise dilute strain and have been estimated to occur at a rate of 3.9 x 10-6 events per gamete, a rate much higher than other recessive coat-color mutations tested (2). Somatic reversions of d have not been reported previously.The unusually high germ-line reverse mutation rate of the d mutation carried by DBA can be explained by the discovery that this mutant allele was generated by the integration of a mobile element into the d gene (3, 4). The transposon has been identified as an ecotropic murine leukemia virus (MuLV) provirus, termed Emv-3 (5). Reversion of d to d+ was correlated with loss of most but not all of the provirus (3, 4). Molecular cloning and DNA sequence analysis of two revertant sites revealed that exactly one viral long terminal repeat (LTR) remained in each revertant chromosome (4, 6). Since revertant animals are wild type in coat color yet carry an LTR at the d locus, it is likely that the Emv-3 provirus induced the dilute mutation by inserting into a noncoding region of the d gene. DNA sequence analysis of the area immediately flanking the proviral integration site showed no significant open reading frames within 600 bases (6), supp...