International Mammalian Genome Society

The 13th International Mouse Genome Conference
October 31-November 3, 1999

Table of Contents * Structure * Bioinformatics * Sequence * Mapping * New Tools * Gene Discovery * Developmental * Mutagenesis * Functional Genomics

I1 An Allelic Series of Mutations at the Steel Locus

M. A. Bedell1, R. Sripriya1, W. S. Davis1, H. K. Jimenez1, D. Willhite1 and L. B. Russell2. 1Department of Genetics, University of Georgia, Athens, GA; 2Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN

The Steel (Sl) locus encodes mast cell growth factor (Mgf) that is the ligand for the Kit receptor tyrosine kinase and is required for multiple steps in the development of germ cells, erythroid cells, mast cells and melanocytes in mice. Two biologically active isoforms of Mgf are produced; a 165 aa soluble isoform (S-Mgf) that is processed from a 248 aa precursor and 220 aa membrane-bound isoform (MB-Mgf) that is expressed from an alternatively spliced transcript. Both S- and MB-Mgf contain four conserved a-helical domains that are thought to mediate folding into a structure known as a four-helix bundle. A valuable genetic resource for studying the role of Mgf in development is a large collection of Sl mutant alleles in the mouse. Previously characterized Sl alleles include a number of deficiencies that contain complete deletions of Mgf coding sequences (e.g. Slgb), two alleles that affect entire domains of the protein (Sld and Sl17H) and two alleles that affect expression of Mgf (Slpan and Slcon). We have now identified intragenic mutations in Mgf sequences of nine new Sl alleles. Seven ethylnitrosourea (ENU)-induced alleles were found to contain point mutations, of which five result in missense mutations. For each of these missense mutations, the allele and the affected amino acids are as follows: Sl195ENURgg, L18R; Sl19ENURc, P23L; Sl2PENURageII, L54P; Sl10ENURc, I118N; and Sl1ZENUe, S122F. Significantly, each of these missense mutations affects sequences within the predicted a-helical domains. Two other ENU-induced Sl alleles were found to contain point mutations. The Sl18ENURk allele contains a nonsense mutation in the extracellular domain of Mgf and is predicted to encode two S-Mgf isoforms with C-terminal deletions. The Sl114ENURd contains a mutation in a 5' splice site and the resulting 8 bp insertion in the cDNA sequence is predicted to cause translation termination after codon 96. In addition to these ENU-induced alleles, we have also identified mutations in two spontaneous Sl alleles. The Sl12HD allele contains a nonsense mutation that is predicted to result in translation termination after only 46 aa. The Sl5CCd allele contains a deletion of 6 nucleotides that is predicted to result in deletion of aa 31 and 32 of Mgf. Both of these spontaneous mutations occur at residues located between the 1st and 2nd a-helical domains. We have determined the effects of each of these nine mutations on viability and anemia in homozygous mice. The results indicate that three of the missense alleles (Sl195ENURgg, Sl19ENURc and Sl10ENURc) and Sl114ENUR behave as null alleles with respect to viability and anemia. However Sl2PENURageII, Sl1ZENUe, Sl18ENURk are hypomorphic with Sl2PENURageII having the most severe effect and Sl18ENURk having the mildest effect. Curiously, Sl18ENURk/Sl18ENURk mice are less anemic than Sld/Sld mice, even though the former mutation is predicted to remove more C-terminal sequences than the latter. Furthermore, Sl1ZENUe /Sl1ZENUe mice are nearly as anemic as Sld/Sld mice, yet the germ cell defects in the former are the mildest of all Sl alleles (see abstract by A. Mahakali et al). By analogy to structure-function analyses with structurally similar cytokines, several of these Sl missense mutations may be expected to interfere with binding to Kit. These mutations should prove useful for gaining a more detailed understanding of in vivo structure-function relationships of Mgf.

Research sponsored by the National Science Foundation, under grant number IBN-9728428, and the University of Georgia Research Foundation.


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