A genome wide screen of 185 G3 pedigrees of ENU mutagenized C57BL/6 mice for defects in T and B cell production by members of Prof Chris Goodnow's laboratory yielded a heritable mutant in every 12th pedigree. Each mutant has been mapped and the causal mutation identified by sequencing cDNAs encoded within the mapped interval. Because there is no ascertainment bias in this set, and the new variants can be compared with a large set of genes known to be required for T cell production, the spectrum of sequence variants provides a unique analysis of the relationship between genes, proteins and mammalian traits.

1. All of the mouse sequence variants alter protein-coding elements, ~75% by direct codon substitution and ~25% via errors in mRNA splicing. The absence of functional sequence variants involving non-coding conservons is striking, and can be interpreted either as indicating these sequences do not serve a critical function in immune cell differentiation, or that they are essential for embryonic development and thus fail to be detected in screens of adult animals.
   
2. Many of the variants are single amino acid mis-sense mutations in T cell genes for which data exists on the effects of complete protein-null alleles. In the case of the transcription factor, Ikaros, the mis-sense allele reveals a much wider role for the protein than inferred from the null, probably because the mis-sense protein still fills its niche in normal multi-protein complexes in the cell and avoids masking of its function by related proteins that fill-in when the protein is absent. In the case of the cell signalling scaffold, Carma-1/Card-11, the substitution in one protein-protein interaction domain dissects discrete functions of the scaffold by disrupting lymphocyte activation by the B cell antigen receptor and CD28 while preserving the response to Toll like receptors and T cell antigen receptor. Dissection of different functions has also been revealed by missense alleles in Zap-70 and JAK3 tyrosine kinases, and a protein-lowering (hypomorphic) allele of the signalling adaptor protein SLP76.
3. A completely novel protein coding gene was identified that is required for T cell differentiation in the thymus. Dr Aude Fahrer.s laboratory in the ANU Division of Biochemistry and Molecular Biology has shown that the protein is ubiquitously expressed and has no identifiable domain motifs, but could be confirmed as the causal sequence variant by complementing the T cell defect in mutant bone marrow by retroviral transduction of wild-type cDNA.

Nelms, K. & Goodnow, C.C. (2001)
Genome-wide mutagenesis with ENU to discover immune regulators.
Immunity 15, 409-418.

Miosge LA, Blasioli J, Blery M, Goodnow CC
Analysis of an ethylnitrosourea-generated mouse mutation defines a cell intrinsic role of nuclear factor kappaB2 in regulating circulating B cell numbers.
J Exp Med. 196:1113-9.

Loy AL, Goodnow CC. (2002)
Novel approaches for identifying genes regulating lymphocyte development and function.
Curr Opin Immunol. 14:260-5.

Jun JE, Wilson LE, Vinuesa CG, Lesage S, Blery M, Miosge LA, Cook MC, Kucharska EM, Hara H, Penninger JM, Domashenz H, Hong NA, Glynne RJ, Nelms KA & Goodnow CC (2003).
Identifying the MAGUK protein Carma-1 as a central regulator of humoral immune responses and atopy by genome wide mouse mutagenesis.
Immunity 18:751-762.

Papathanasiou P, Perkins AC, Cobb BS, Ferrini R, Sridharan R, Hoyne GF, Nelms KA, Smale ST & Goodnow CC (2003)
Widespread failure of hematolymphoid differentiation caused by a point mutant allele of the Ikaros transcription factor.
Immunity 19:131-144.

Jun JE, Goodnow CC
Scaffolding of antigen receptors for immunogenic versus tolerogenic signaling.
Nature Immunol. 2003;4(11):1057-64.