Cloning and characterization of the Drosophila homolog of the xeroderma pigmentosum complementation-group B correcting gene, ERCC3.

Previously the human nucleotide excision repair gene ERCC3 was shown to be responsible for a rare combination of the autosomal recessive DNA repair disorders xeroderma pigmentosum (complementation group B) and Cockayne's syndrome (complementation group C). The human and mouse ERCC3 proteins contain several sequence motifs suggesting that it is a nucleic acid or chromatin binding helicase. To study the significance of these domains and the overall evolutionary conservation of the gene, the homolog from Drosophila melanogaster was isolated by low stringency hybridizations using two flanking probes of the human ERCC3 cDNA. The flanking probe strategy selects for long stretches of nucleotide sequence homology, and avoids isolation of small regions with fortuitous homology. In situ hybridization localized the gene onto chromosome III 67E3/4, a region devoid of known D.melanogaster mutagen sensitive mutants. Northern blot analysis showed that the gene is continuously expressed in all stages of fly development. A slight increase (2-3 times) of ERCC3Dm transcript was observed in the later stages. Two almost full length cDNAs were isolated, which have different 5' untranslated regions (UTR). The SD4 cDNA harbours only one long open reading frame (ORF) coding for ERCC3Dm. Another clone (SD2), however, has the potential to encode two proteins: a 170 amino acids polypeptide starting at the optimal first ATG has no detectable homology with any other proteins currently in the data bases, and another ORF beginning at the suboptimal second startcodon which is identical to that of SD4. Comparison of the encoded ERCC3Dm protein with the homologous proteins of mouse and man shows a strong amino acid conservation (71% identity), especially in the postulated DNA binding region and seven 'helicase' domains. The ERCC3Dm sequence is fully consistent with the presumed functions and the high conservation of these regions strengthens their functional significance. Microinjection and DNA transfection of ERCC3Dm into human xeroderma pigmentosum (c.g. B) fibroblasts and group 3 rodent mutants did not yield detectable correction. One of the possibilities to explain these negative findings is that the D.melanogaster protein may be unable to function in a mammalian repair context

Female receptivity phenotype of icebox mutants caused by a mutation in the L1-type cell adhesion molecule neuroglian

Relatively little is known about the genes and brain structures that enable virgin female Drosophila to make the decision to mate or not. Classical genetic approaches have identified several mutant females that have a reluctance-to-mate phenotype, but most of these have additional behavioral defects. However, the icebox (ibx) mutation was previously reported to lower the sexual receptivity of females, without apparently affecting any other aspect of female behavior. We have shown that the ibx mutation maps to the 7F region of the Drosophila X chromosome to form a complex complementation group with both lethal and viable alleles of neuroglian(nrg). The L1-type cell adhesion molecule encoded by nrg consists of six immunoglobulin-like domains, five fibronectin-like domains, one transmembrane domain and one alternatively spliced intracellular domain. The ibx strain has a missense mutation causing a glycine-to-arginine change at amino acid 92 in the first immunoglobulin domain of nrg. Defects in the central brain of ibx mutants are similar to those observed in another nrg mutant, central brain deranged1 (ceb1). However, both ceb1 homozygous and ceb1/ibx heterozygous females are receptive. The expression of a transgene containing the non-neural isoform of nrg rescues both the receptivity and the brain structure phenotypes of ibx females