Molecular analysis of two mouse dilute locus deletion mutations: spontaneous dilute lethal20J and radiation-induced dilute prenatal lethal Aa2 alleles.

Abstract

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...

Rab27a Is an Essential Component of Melanosome Receptor for Myosin Va

“…l20J (Strobel et al, 1990;Wu et al 1997 (Wu et al, 1998). These primary cultures were prepared from the skin of newborn mice as described previously , and they were grown on gelatin-coated Labtek chamber slides with coverglass bottoms (#155380; Nalge Nunc International, Naperville, IL) or #1.5 round coverslips in Ham's F-10 medium supplemented with 10% horse serum, 2% fetal calf serum, 1% penicillin streptomycin, 0.1 M dibutryl cAMP, and 85 nM phorbol 12-myristate 13-acetate (#P-8139; Sigma-Aldrich, St. Louis, MO).…”

“…To accomplish this, we microinjected primary melanocytes cultured from the skin of newborn mice homozygous for a true null allele at dilute (d l20J ) (Strobel et al, 1990;Wu et al, 1997) with plasmids encoding various full- (Figure 6, J-L), which lack both exons D and F, did not rescue a single mutant cell (n ϭ 74 and 72 for MC MV⌬F and BR MV, respectively) (given that the fluorescence signal from these GFP-tagged myosins, in contrast to the signal from the GFP-tagged dominant negative constructs, was often very weak, the melanocytes in these experiments were coinjected with plasmid EGFP C1, which allowed for unequivocal identification of all microinjected cells, and, therefore, accurate scoring of rescue; the fluorescence images in C, F, I, and L reveal primarily the distribution of the unfused GFP expressed from this plasmid). Furthermore, in dilute melanocytes that were injected with just the plasmids encoding GFP-tagged myosin Va isoforms, and that happened to exhibit a strong fluorescence signal, myosin MC MV (Figure 7, A1-B2) and MC MV⌬D (Figure 7, C and D, plus inset) showed extensive colocalization with black, end-stage organelles in rescued cells, whereas MC MV⌬F ( Figure 7, E and F) and BR MV (Figure 7,G and H) showed no tendency to associate with the melanosomes clustered in the perinuclear region.…”

“…Melanocytes generate this peripheral accumulation of melanosomes via a cooperative transport mechanism in which fast, long-range, bidirectional, microtubule-dependent melanosome movements along the length of dendrites are coupled to myosin Va-dependent capture and local movement of the organelles within distal, actin-rich regions of the dendrite (Wu et al, 1998). When the capture mechanism is missing, as in melanocytes from mice homozygous for a functional null allele at dilute (d l20J ) (Strobel et al, 1990), melanosomes redistribute according to microtubule density, leading to their accumulation in the central cytoplasm where the bulk of microtubules reside (Koyama and Takeuchi, 1981;Provance et al, 1996;Wei et al, 1997;Wu et al, 1998). Long-range, microtubule-based melanosome movements within dendrites continue in the absence of myosin Va, but the bidirectional nature of these movements prevents them from generating a peripheral accumulation of the organelles on their own (Wu et al, 1998).…”

Why Genetic Effects of Radiation are Observed in Mice but not in Humans

“…This difference may be reduced to one-half if one assumes that nearly one-half of the induced deletions were too small to be detectable with the array-CGH method. However, the difference cannot be further reduced by assuming that small mutations were the predominant-type of deletions, because many mutagenesis studies in cultured mammalian cells at the HPRT gene [e.g., see (39)] and also in germ cells of mice [e.g., see (5,40,41)] have consistently indicated that large deletions are the predominant type of mutations induced by radiation.…”

“…However, one mutant allele (d l20J ) at the d locus (one of the specific locus genes used in the SLT), which was known to cause homozygous lethality, was found to be an intralocus deletion (41); also, an artificially knocked-out null allele of the s gene (another gene used for SLT studies) caused homozygous lethality (46). These results raised the question of why the mutant homozygous animals used for the tester strain were viable and fertile.…”