Molecular Basis Of Wand Sl Phenotypes

Numerous different W and Sl mutant mice have been studied, and many of the underlying mutations have now been characterized at the molecular level (6). W, the first "dominant white spotting" mutant described (in the early 1900s) (2), is now known to represent a null mutation affecting c-kit mRNA splicing such that the c-kit polypeptide lacks a transmembrane domain, is not expressed at the cell surface, and lacks kinase activity (4,6). Homozygous animals are severely affected with respect to hematopoiesis, melanogenesis, and gametogenesis and die within a week after birth (1,2). Heterozygotes have some white spotting and coat color dilution but otherwise are relatively normal, and fertile. Therefore the W locus is semidominant. The W (W-viable) mutation represents a Thr660 ^ Met change in the first kinase homology region and confers reduced kinase activity (4,6). Homozygous (WW) animals are affected almost as severely as W/W animals, and most die within 3 wk after birth (1,2). Heterozygotes (Wv/+) are affected somewhat more so than W/+ heterozygotes with respect to all pheno-types, i.e., this mutation is dominant negative, probably as a result of an inhibitory effect of Wv monomers dimerizing with wild-type c-kit monomers. Mice of the compound heterozygous genotype WW are the most studied and most used W mutants; they survive for a considerable period after birth and display all the characteristic phenotypes to a considerable extent (including sterility). W/Wv mice are generated by crosses of W/+ mice and W/+ mice, both being viable and fertile. A fairly accurate generalization with respect to W mutations is that the extent of phenotypic defects correlates with the extent to which kinase activity is diminished (6). Activating c-kit mutations have been described (see also Subheading 13).

Sl was the first "steel" mutant, named and described in 1956 (3). It is now known to be a null mutation, with the entire locus deleted (4,6). Homozygous animals are severely affected with respect to hematopoiesis, melanogenesis, and gametogenesis and die in utero (1,3). Heterozygotes (Sl/+) have a moderate anemia plus some white spotting and coat color dilution but otherwise are relatively normal, and fertile. Therefore the Sl locus, like W, is semidominant.

The Sld (Sl-Dickie) mutation represents an intragenic deletion in the Sl gene such that a polypeptide of 183 amino acids (not counting the signal sequence) is made; the first 180 amino acids correspond to wild type, and the last 3 are novel (4,6,7). This gene product lacks the transmembrane and cytoplasmic domains and consequently is secretable/soluble. The basolateral cell surface targeting sequences normally present in the cytoplasmic portion of SCF are of course missing. Homozygous (Sld/Sld) animals have a moderate macrocytic anemia, lack coat color, and are sterile but can be viable for up to 1 yr (1,3). Heterozygotes (Sld/+) are similar to Sl/+ animals with respect to all phenotypes. Mice of the compound heterozygous genotype Sl/Sld are probably the most studied of Sl mutants; they survive for up to 1 yr after birth, with severe macro-

cytic anemia, lack of coat color, and severe germ cell deficiency/sterility. Sl/Sld mice are generated by crosses of Sl/+ mice and Sld/+ mice, both of which are fertile. Amounts of SCF protein in Sl/Sld mice appear to be reduced to a much greater extent than would be expected from the gene dosage effect alone (35,36).

Analysis of Sl17H mutant mice suggested some importance for the cytoplasmic portion of membrane-associated SCF. The Sl17H allele is missing exon 8 (which encodes most of the 35-amino acid cytoplasmic portion) with a frameshift such that the resulting polypeptide has a 27-amino acid cytoplasmic portion of unique sequence (4,6,7). Homozygotes (Sl17H/Sl17H) are affected (relatively mildly) in the hematopoiesis, melanocyte, and germ cell lineages. Based on in vitro studies of cells transfected with Sl17H, expression of SCF protein at the cell surface is at most half of normal; dimeriza-tion of the cell surface SCF is decreased; and reasonably normal amounts of soluble SCF are released (4,6,7,19,24,37). The basolateral-cell-surface-targeting sequences are altered, and an endocytosis-targeting sequence (in the unique cytoplasmic portion) may contribute to the decreased cell surface expression (19,20).

Homozygous Sl17H/Sl17H mice, in which males are sterile whereas females are fertile, also represent an example of differential mutational effects in different affected tissues— a phenomenon that is quite common among the many W and Sl mutants (4-7). In the case of Sl17H/Sl17H, it may be that basolateral localization of SCF (on Sertoli cells and on epithelial cells) is particularly important within the cellular and tissue architecture of the testis and skin (respectively), where SCF-dependent spermatocytes and melanocytes (respectively) are developing, and less important for hematopoiesis in bone marrow and spleen or for oogenesis in the ovary (20). For other mutations, the phenomenon may reflect differences between the various tissues in the functional dependence on SCF/c-kit; or alterations (e.g., in upstream regulatory sequences) that affect expression in some tissues more so than in others (6).

Getting Back Into Shape After The Pregnancy

Getting Back Into Shape After The Pregnancy

Once your pregnancy is over and done with, your baby is happily in your arms, and youre headed back home from the hospital, youll begin to realize that things have only just begun. Over the next few days, weeks, and months, youre going to increasingly notice that your entire life has changed in more ways than you could ever imagine.

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