MOLECULAR AND GENETIC CHARACTERISATION OF THE EAST GENE OF DROSOPHILA MELANOGASTER

Abstract

This thesis describes the molecular and genetic characterisation of the east (enhanced adult sensory threshold) gene of Drosophila melanogaster. east was identified in a transposon mediated insertional mutagenesis screen for mutants impaired in their behavioural responses to chemosensory stimuli. The insertion of a P-element (a Drosop hila transposon) into the east locus on the X-chromosome results in reduced responses of adult flies to olfactory and gustatory stimuli. In addition, the east mutation affects the electrophysiology of peripheral chemosensory neurons. A precise excision of the P-element can reconstitute wildtype behaviour, demonstrating that the inserted transposon is indeed responsible for the mutant phenotype. The east gene encodes at least 4 transcripts generated by alternative splicing and promoter usage. The expression of these mRNAs, which have estimated sizes of 1.6, 2.9, 8. l and 10.4 kb, is subject to developmental and tissue specific regulation. The cDNA corresponding to the 8. l kb transcript was cloned and sequenced. It encodes a predicted open reading frame of 2362 amino acids containing 6 putative nuclear localisation sequences (NLS) and 12 potential PEST sites. The EAST protein shares no homology with other sequences from the database. Antibodies were raised against 4 different portions of the EAST protein. Consistent with the presence of NLS sites, all antisera detected a ubiquitously expressed nuclear protein. Immunolocalisation and Western blotting studies indicate that the EAST protein is associated with the nuclear matrix, the structural framework of the nucleus consisting of ribonucleoproteins. In the polyploid interphase nuclei of 3rd instar larval salivary glands, EAST is localised to the space between the chromosomes. Fractionation studies of embryonic nuclei showed that EAST is associated with the nuclear matrix but not with the chromatin fraction. Consistent with the presence of 12 potential PEST sites, which are supposed to target proteins for rapid degradation, EAST shows a rather dynamic expression pattern. In proliferating tissues, the concentration of EAST fluctuates in a cell cycle dependent fashion, with an increase in expression coinciding with both disassembly and reassembly of the interphase nucleus. The homozygous viable east alleles causing behavioural abnormalities are hypomorphs. Stronger deletion alleles leading to lethality indicate that east is required for viability. These lethal mutations disrupt a variety of nuclear and genomic fictions. Female flies heterozygous for a subset of the recessive lethal alleles show defects in the segregation of non-exchange X-chromosomes during meiosis I. Male flies homozygous for one lethal allele were also affected in meiosis. The occurrence of multinuclear spermatids suggests that the spermatocytes are defective in cytokinesis. The phenotypic analysis of east(m) embryos from which maternal east expression was removed provided further insights into the gene function. Although the germline expression of east is not absolutely required for oogenesis and viability, it greatly improves the rate of survival of embryos. During the syncytial blastoderm stage, east(m) embryos show a variety of mitotic defects, including the elimination of nuclei. Other mutant phenotypes such as the a synchronicity of dividing nuclei or formation of bipolar tubulin rings around interphase and prophase nuclei suggest that the removal of maternal east slows down the entry into and the exit from mitosis. east also seems to be involved in the maintenance of transcription. Ventral cells of east(m) embryos showed an inactivation of twist gene expression in a random pattern from gastrulation onwards. Extensive rearrangements of chromosomes occurring prior to and after mitosis coincide with phases of strong EAST expression. As a putative component of the nuclear matrix, EAST could play a role in altering or maintaining the spatial organisation of the chromosomes.