Institut fuer Allgemeine Genetik (240), Universitaet Hohenheim, D- 70593 Stuttgart, F.R.G., § Genetics Department, University of Glasgow, Scottland, UK,
Enhancer of split [E(spl)] is a gene complex comprising seven bHLH genes and gro, all thought to be integral part in the N signalling pathway. Originally, E(spl) was identified by a dominant allele, that specifically enhances the recessive split allele of the Notch locus. The dominant E(spl) allele is homozygous viable, however, it is lethal in hemizygosis if the deficient chromosome is provided by the mother. Thus, this mutation displays two distinct phenotypes. In accordance, two molecular lesions are found: a 6.4 kb insertion in the third intron of the gro gene and a small deletion truncating the adjacent m8 bHLH transcript at the 3' end (m8*). The large insertion has no apparent effect on gro transcription in the embryo, nor on gro protein distribution during eye imaginal discs development and during oogenesis, respectively. Whilst the hemizygous lethality can be rescued by additional wild type gro gene copies, the gro gene of the mutant has lost this activity, as well as the ability to rescue gro point mutations. The mutation in m8* is the basis of the above described phenotypic interactions, since m8* alone, provided as a transgene, will enhance the split phenotype in an otherwise wild type background. As a consequence of the truncation, the m8* mRNA is apparently stabilized and a frame shift alters the important WRPW C-terminus of the m8* protein. Normally, this C-terminus of the m8 bHLH protein is bound by the gro protein. The ability of gro to bind m8* despite the alterations is currently investigated using the yeast-2-hybrid system. Molecular analyses of three revertants indicates that the m8* truncation is not the sole cause of the phenotype but also the newly acquired amino acids. To test this, hybrid constructs were created, where the C-terminal halfs of E(spl) bHLH proteins m5 and md, respectively, were exchanged by mutant m8*. No effect was seen with m5-m8*. This might have been expected since this construct was under m5 control, and m5 gene expression in the eye disc is barely detectable. In agreement with this interpretation, the same construct under m8 control enhances the split phenotype. A much stronger effect was seen with the md-m8* hybrid, in accordance with the strong md expression in the eye disc. These results provide evidence that the altered m8* C-teminus is directly responsible for split enhancement, influenced by the total amount of the mutant product. Apparently, the m8 bHLH as well as the HEL (orange) domain can be replaced by other E(spl) proteins, supporting the idea of functional redundancy. Surprisingly enough, even though m5 and m8 are the most closely related of the E(spl) bHLH genes, this hybrid has the least impact, suggesting distinct, separable functions of the respective proteins.