Comparative analysis of circadian clock genes in insects.

After a slow start, the comparative analysis of clock genes in insects has developed into a mature area of study in recent years. Brain transplant or surgical interventions in larger insects defined much of the early work in this area, before the cloning of clock genes became possible. We discuss the evolution of clock genes, their key sequence differences, and their likely modes of regulation in several different insect orders. We also present their expression patterns in the brain, focusing particularly on Diptera, Lepidoptera, and Orthoptera, the most common non-genetic model insects studied. We also highlight the adaptive involvement of clock molecules in other complex phenotypes which require biological timing, such as social behaviour, diapause and migration.

Efficient and heritable functional knock-out of an adult phenotype in Drosophila using a GAL4-driven hairpin RNA incorporating a heterologous spacer.

We have developed a modified RNA interference (RNAi) method for generating gene knock-outs in Drosophila melanogaster. We used the sequence of the yellow (y) locus to construct an inverted repeat that will form a double-stranded hairpin structure (y-IR) that is under the control of the upstream activating sequence (UAS) of the yeast transcriptional activator GAL4. Hairpins are extremely difficult to manipulate in Escherichia coli, so our method makes use of a heterologous 330 bp spacer encoding sequences from green fluorescent protein to facilitate the cloning steps. When the UAS-y-IR hairpin is expressed under the control of different promoter-GAL4 fusions, a high frequency of y pigment phenocopies is obtained in adults. Consequently this method for producing gene knock-outs has several advantages over previous methods in that it is applicable to any gene within the fly genome, greatly facilitates cloning of the hairpin, can be used if required with GAL4 drivers to avoid lethality or to induce RNAi in a specific developmental stage and/or tissue, is useful for generating knock-outs of adult phenotypes as reported here and, finally, the system can be manipulated to investigate the trans-acting factors that are involved in the RNAi mechanism.

Comparative analysis of the nonA region in Drosophila identifies a highly diverged 5' gene that may constrain nonA promoter evolution.

A genomic fragment from Drosophila virilis that contained all the no-on-transientA (nonA) coding information, plus several kilobases of upstream material, was identified. Comparisons of nonA sequences and the gene nonA-like in D. melanogaster, a processed duplication of nonA, suggest that it arose before the split between D. melanogaster and D. virilis. In both species, another gene that lies <350 bp upstream from the nonA transcription starts, and that probably corresponds to the lethal gene l(1)i19, was identified. This gene encodes a protein that shows similarities to GPI1, which is required for the biosynthesis of glycosylphosphatidylinositol (GPI), a component for anchoring eukaryotic proteins to membranes, and so we have named it dGpi1. The molecular evolution of nonA and dGpi1 sequences show remarkable differences, with the latter revealing a level of amino acid divergence that is as high as that of transformer and with extremely low levels of codon bias. Nevertheless, in D. melanogaster hosts, the D. virilis fragment rescues the lethality associated with a mutation of l(1)i19e, as well as the viability and visual defects produced by deletion of nonA(-). The presence of dGpi1 sequences so close to nonA appears to have constrained the evolution of the nonA promoter.

Molecular Dissection of the 5' Region of no-on-transientA of Drosophila melanogaster Reveals cis-Regulation by Adjacent dGpi1 Sequences.

The nonA gene of Drosophila melanogaster is important for normal vision, courtship song, and viability and lies approximately 350 bp downstream of the dGpi1 gene. Full rescue of nonA mutant phenotypes can be achieved by transformation with a genomic clone that carries approximately 2 kb of 5' regulatory material and that encodes most of the coding sequence of dGpi1. We have analyzed this 5' region by making a series of deleted fragments, fusing them to yeast GAL4 sequences, and driving UAS-nonA expression in a mutant nonA background. Regions that both silence and enhance developmental tissue-specific expression of nonA and that are necessary for generating optomotor visual responses are identified. Some of these overlap the dGpi1 sequences, revealing cis-regulation by neighboring gene sequences. The largest 5' fragment was unable to rescue the normal electroretinogram (ERG) consistently, and no rescue at all was observed for the courtship song phenotype. We suggest that sequences within the nonA introns that were missing in the UAS-nonA cDNA may carry enhancer elements for these two phenotypes. Finally, we speculate on the striking observation that some of the cis-regulatory regions of nonA appear to be embedded within the coding regions of dGpi1.

Conceptual translation of timeless reveals alternative initiating methionines in Drosophila.

We have sequenced genomic fragments which encode the N-terminus of the TIMELESS (TIM) clock protein in Drosophila simulans and D. yakuba. We observe that in these two species, the initiating methionine appears to lie downstream of the one proposed to encode the translational start inD.melanogaster, thereby truncating the N-terminus by 23 amino acids. We then sequenced the corresponding 5'fragment in a number of D. melanogaster individuals from different strains. We observed a polymorphism which strongly suggests that the originally proposed start site cannot be utilised in some individuals, and that these flies will initiate translation of TIM at the downstream ATG. Given the current interest in TIM regulation in D. melanogaster, it is important to correctly define the N-terminus in this species.

Cytogenetic and immunofluorescence analysis of benzo[a]pyrene-DNA adduct formation and chromosome damage in larval brain neuroblasts of Drosophila melanogaster.

Recently we have evaluated the relationship between benzo[a]-pyrene(BaP)-DNA adducts, determined by 32P-postlabelling, and clone frequencies in the somatic mutation and recombination test (SMART) in Drosophila melanogaster. Following that study we proceeded to characterise further the mechanism of induction of genetic damage in vivo by BaP in Drosophila by cytogenetic analysis of larval brain neuroblasts. Third stage larvae were treated with 4 and 10 mM BaP for 24, 48 or 72 h. In all cases, the larvae were killed 72 h after the beginning of treatment, entailing 48, 24 or 0 h post-treatment recovery in BaP-free medium, respectively. At the end of the treatment the following data were collected: (i) the types and levels of chromosome aberrations in neuroblast metaphase and anaphase nuclei; (ii) the distribution and level of BaP-DNA adducts, revealed by indirect immunofluorescence in neuroblast nuclei using an anti-(BaP-DNA) antibody. The results indicate that BaP induces chromosome breaks, deletions and exchanges in this system. In particular, chromosome exchanges decrease as the post-treatment recovery time increases, and the dynamics of breaks and deletions appear to be inversely related to those of the exchanges. This suggests that exchanges may require few preconditions to occur and are thus expressed soon after treatment. Chromosome breaks and deletions could require multiple single events before the actual damage is expressed (even some cell divisions away from the end of treatment). The immunofluorescence analysis suggests that BaP-DNA adducts are more abundant in the heterochromatin of the neuroblast nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)