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To understand the role of a gene in adult behavior, it is necessary to control its expression in four dimensions: space and time. Two recent papers describe implementation of different but related technologies that now provide this missing element in fly behavioral research.
We have reported that several silkworm strains are permissive to intrahemocoelical infection of Autographa californica nucleopolyhedrovirus (AcNPV), contrary to the general belief that AcNPV cannot infect silkworm. In the present study, we address whether the intrahemocoelical infection of AcNPV to the silkworm was an exceptional phenomenon, and the possible genetic basis underlying it. Wilder range test of 31 strains of silkworm Bombyx mori for intrahemocoelical AcNPV infection led to the identification of 14 permissive strains and 17 nonpermissive strains, indicating that the intrahemocoelical infection of AcNPV to the silkworm was not a rare and isolated phenomenon. Productive infection was shown in permissive silkworms, by EGFP fluorescence in various tissues when expression of reporter gene controlled by a very late viral promoter polh. The viral titer in larval hemolymph of permissive silkworms increased and maintained at a higher level hundredfold more than the initial amount of virus, indicating viral replication. A series of genetic cross experiments suggested the existence of only one dominant host anti-AcNPV gene or a set of genetically linked genes, which prevent AcNPV infection in nonpermissive silkworm strain Qingsong and are absent in permissive silkworm strain Haoyue.
Previous estimates of a generic level phylogeny for the ubiquitous parasitoid wasp subfamily Microgastrinae (Hymenoptera) have been problematic due to short internal branches deep in the phylogeny. These short branches might be attributed to a rapid radiation among the taxa, the use of genes that are unsuitable for the levels of divergence being examined, or insufficient quantity of data. We added over 1200 nucleotides from four nuclear genes to a dataset derived from three genes to produce a dataset of over 3000 nucleotides per taxon. While the number of well-supported short branches in the phylogeny increased, we still did not obtain strong bootstrap support for every node. Parametric and nonparametric bootstrap simulations projected that an enormous, and likely unobtainable, amount of data would be required to get bootstrap support greater than 50% for every node. However, a marked increase in the number of well-supported nodes was seen when we conducted a Bayesian analysis of a combined dataset generated from morphological characters added to the seven gene dataset. Our results suggest that, in some cases, combining morphological and genetic characters may be the most practical way to increase support for short branches deep in a phylogeny.
During the development of the Drosophila wing, the activity of the Notch signalling pathway is required to establish and maintain the organizing activity at the dorsoventral boundary (D/V boundary). At early stages, the activity of the pathway is restricted to a small stripe straddling the D/V boundary, and the establishment of this activity domain requires the secreted molecule fringe (fng). The activity domain will be established symmetrically at each side of the boundary of Fng-expressing and non-expressing cells. Here, I present evidence that the Drosophila tumour-suppressor gene lethal (2) gaint discs (lgd) is required to restrict the activity of Notch to the D/V boundary. In the absence of lgd function, the activity of Notch expands from its initial domain at the D/V boundary. This expansion requires the presence of at least one of the Notch ligands, which can activate Notch more efficiently in the mutants. The results further suggest that Lgd appears to act as a general repressor of Notch activity, because it also affects vein, eye, and bristle development.
We searched for the previously defined uncoupling protein (UCP) signatures [Jezek, P. and Urbánková, E. (2000) IUBMB Life 49, 63-70] in genomes of Drosophila melanogaster, Caenorhabditis elegans, Dictyostelium discoideum, and Arabidopsis thaliana. We identified four UCPs in Drosophila and one in Caenorhabditis or Dictyostelium as close relatives of human UCP4 (BMCP), but distant from UCP1, UCP2, UCP3, and two plant UCPs of Arabidopsis. But the third Arabidopsis UCP is the closest UCP4 relative. This suggests that UCP4 represents the ancestral UCP from which other mammalian and plant UCPs diverged. Speculations on UCP4 participation in apoptosis are thus supported by its early phylogenetic occurrence.
We investigated the functions of clock genes period (per) and timeless (tim) in establishing negative feedback on circadian transcription factors clock/cycle (Clk/cyc) in Drosophila. We show that PER protein persists for several hours after rapid degradation of TIM in the morning. We observed in cell culture that isolated PER inhibits CLK/CYC-activated transcription in the absence of TIM and we further demonstrated for the first time in vivo that PER accumulation in a tim loss-of-function mutant background causes efficient inhibition of CLK/CYC-dependent transcription. These results identify PER to be the main inhibitor for CLK/CYC and they suggest a delay mechanism during early morning, when PER protein, after degradation of TIM, forms an inhibitor buffer for CLK/CYC that attenuates the restart of the next cycle of CLK/CYC-activated transcription. While TIM likely enhances the inhibition of CLK/CYC by PER in the dark, our results suggest a reduction of PER-mediated inhibition by TIM in light.
Splicing enhancers have previously been shown to promote processing of introns containing weak splicing signals. Here, we extend these studies by showing that also 'strong' constitutively active introns are absolutely dependent on a downstream splicing enhancer for activity in vitro. SR protein binding to exonic enhancer elements or U1 snRNP binding to a downstream 5' splice site serve redundant functions as activators of splicing. We further show that a 5' splice site is most effective as an enhancer of splicing. Thus, a 5' splice site is functional in S100 extracts, under conditions where a SR enhancer is nonfunctional. Also, splice site pairing occurs efficiently in the absence of exonic SR enhancers, emphasizing the significance of a downstream 5' splice site as the enhancer element in vertebrate splicing.
The chaperonins are evolutionarily conserved essential cellular proteins that help folding newly synthesized or translocated proteins, spending ATP. We present here the molecular analysis of the hsp60 gene promoter region and of two Drosophila hsp60 ethyl methane sulfonate embryonic lethal alleles that have an identical phenotype. No heat shock element sequences were found in the 5' region, supporting previous data (Kozlova, T. et al., 1997) which suggests that mitochondrial Drosophila melanogaster HSP60.1 is not heat inducible. By sequencing the lethal allele's entire open reading frame (ORF), we found a C-T transition in the hsp60F409 allele that produces a serine to leucine change, apparently distorting the protein equatorial domain structure. No changes were found in the hsp60G93 ORF. However, an analysis of the heterogeneous nuclear RNA levels showed a reduction of the hsp60 transcript in hsp60G93 flies as compared to the wild-type. These data suggest that although the defects in the hsp60 gene produced by these alleles are at different levels, both behave as null mutations.
Expression of the Drosophila segment polarity genes is initiated by a pre-pattern of pair-rule gene products and maintained by a network of regulatory interactions throughout several stages of embryonic development. Analysis of a model of gene interactions based on differential equations showed that wild-type expression patterns of these genes can be obtained for a wide range of kinetic parameters, which suggests that the steady states are determined by the topology of the network and the type of regulatory interactions between components, not the detailed form of the rate laws. To investigate this, we propose and analyse a Boolean model of this network which is based on a binary ON/OFF representation of mRNA and protein levels, and in which the interactions are formulated as logical functions. In this model the spatial and temporal patterns of gene expression are determined by the topology of the network and whether components are present or absent, rather than the absolute levels of the mRNAs and proteins and the functional details of their interactions. The model is able to reproduce the wild-type gene expression patterns, as well as the ectopic expression patterns observed in overexpression experiments and various mutants. Furthermore, we compute explicitly all steady states of the network and identify the basin of attraction of each steady state. The model gives important insights into the functioning of the segment polarity gene network, such as the crucial role of the wingless and sloppy paired genes, and the network's ability to correct errors in the pre-pattern.
The isolation and analysis of mutant flies (Drosophila melanogaster) with altered circadian rhythms have led to an understanding of circadian rhythms at the molecular level. This molecular mechanism elucidated in fruit flies is similar to the mechanism of the human circadian clock, which confers 24-h rhythmicity to our sleep/wake behavior, as well as to many other aspects of our cellular and organismal physiology. In fruit flies, genes can be mutated to abolish circadian rhythms (i.e., produce arrhythmia) or alter the period of the circadian rhythm; these genes encode key components of the circadian oscillator mechanism. Other mutations have identified components of the input pathways (by which light and temperature synchronize the circadian clock to environmental cycles) or output pathways (which connect the circadian oscillator to the physiological response). Mutations in genes are typically generated by chemical mutagenesis or mutagenesis with transposable elements. Flies with mutagenized chromosomes are processed in a series of genetic crosses, which allow specific chromosomes to be screened for semidominant mutations, recessive mutations, enhancer/suppressor mutations, or genes that can be overexpressed to alter circadian rhythms. Circadian phenotypes, which are assayed to identify mutants, include eclosion (emergence of the adult from the pupal case), locomotor activity (similar to human sleep?wake behavior), and circadian oscillations of gene expression. It is argued that screens for new circadian genes will continue to reveal novel components of the circadian mechanism.
The Drosophila melanogaster fuzzy onions (fzo) gene encodes the first known protein mediator of mitochondrial fusion. During Drosophila spermatogenesis, mitochondria in early postmeiotic spermatids aggregate, fuse, and elongate beside the growing flagellar axoneme. fzo mutant males are defective in this developmentally regulated mitochondrial fusion and are sterile. fzo encodes a large, novel, predicted transmembrane GTPase that becomes detectable on spermatid mitochondria late in meiosis II, just prior to fusion, and disappears soon after fusion is complete. Missense mutations that alter conserved residues required for GTP binding in other GTPases inhibit the fusogenic activity of Fzo in vivo but do not affect its localization. Fzo has homologs of unknown function in mammals, nematodes, and yeast.
The steroid hormone ecdysone directs Drosophila metamorphosis via three heterodimeric receptors that differ according to which of three ecdysone receptor isoforms encoded by the EcR gene (EcR-A, EcR-B1, or EcR-B2) is activated by the orphan nuclear receptor USP. We have identified and molecularly mapped two classes of EcR mutations: those specific to EcR-B1 that uncouple metamorphosis, and embryonic-lethal mutations that map to common sequences encoding the DNA- and ligand-binding domains. In the larval salivary gland, loss of EcR-B1 results in loss of activation of ecdysone-induced genes. Comparable transgenic expression of EcR-B1, EcR-B2, and EcR-A in these mutant glands results, respectively, in full, partial, and no repair of that loss.
When two to six copies of a white promoter-Alcohol dehydrogenase (Adh) reporter fusion gene are introduced into the genome, the expression is progressively reduced both in larvae and adults rather than the expected gene dosage effect. In addition, multiple transgenes reduce endogenous Adh transcripts, a result that is strongly analogous to "cosuppression" phenomena described in many plant species but which has not been previously observed in animals. Silencing of the Adh gene is not influenced by zeste-dependent transvection but strongly affected by the Polycomb and Polycomblike mutations. Polycomb and polyhomeotic proteins are bound to the chromatin at the sites of the repressed w-Adh transgenes.
Pulses of ecdysteroids direct Drosophila through its life cycle by activating stage- and tissue-specific genetic regulatory hierarchies. Here we show that an orphan nuclear receptor, DHR78, functions at the top of the ecdysteroid regulatory hierarchies. Null mutations in DHR78 lead to lethality during the third larval instar with defects in ecdysteroid-triggered developmental responses. Consistent with these phenotypes, DHR78 mutants fail to activate the mid-third instar regulatory hierarchy that prepares the animal for metamorphosis. DHR78 protein is bound to many ecdysteroid-regulated puff loci, suggesting that DHR78 directly regulates puff gene expression. In addition, ectopic expression of DHR78 has no effects on development, indicating that its activity is regulated post-translationally. We propose that DHR78 is a ligand-activated receptor that plays a central role in directing the onset of Drosophila metamorphosis.
The coordination between cell proliferation and cell death is essential to maintain homeostasis within multicellular organisms. The mechanisms underlying this regulation are yet to be completely understood. Here, we report the identification of hippo (hpo) as a gene that regulates both cell proliferation and cell death in Drosophila. hpo encodes a Ste-20 family protein kinase that binds to and phosphorylates the tumor suppressor protein Salvador (Sav), which is known to interact with the Warts (Wts) protein kinase. Loss of hpo results in elevated transcription of the cell cycle regulator cyclin E and the cell-death inhibitor diap1, leading to increased proliferation and reduced apoptosis. Further, we show that hpo, sav, and wts define a pathway that regulates diap1 at the transcriptional level. A human homolog of hpo completely rescues the overgrowth phenotype of Drosophila hpo mutants, suggesting that hpo might play a conserved role for growth control in mammals.
Mutations in the human PTEN gene have been identified in a number of different tumour types, and in the hamartomatous polyposis syndromes Cowden disease and Bannayan-Zonana syndrome. The PTEN gene encodes a phosphatase that antagonises phosphoinositide 3-kinase (PI3K) signalling by removing the 3' phosphate from phosphatidylinositol 3, 4,5-trisphosphate (PtdIns (3,4,5)P(3)). Here we show that the PTEN gene is conserved in the invertebrate Drosophila melanogaster and demonstrate that the gene undergoes alternative splicing.
Recessive oncogenes have genetic functions important for the regulation of tissue growth and differentiation. Defining the role of these genes in normal developmental and physiological processes is important to the development of the accurate models of the normal regulation of growth and differentiation. We report here a genetic analysis of the requirement for the lethal (2) giant larva function during development. The results demonstrate that the lethal (2) giant larva function is required during embryonic and post-embryonic development to maintain the normal developmental capacity.
The Drosophila melanogaster homolog of the human proto-oncogene fos is Dfos. It is the only fos homolog in the Drosophila genome. Fos functions as a subunit of the heterodimeric transcription factor AP-1. There are two models of the Dfos gene. The first comes from a cDNA sequence of Dfos (Perkins et al., Genes Dev. 4 (1990) 822). The second is from the gene sequence published by the Drosophila genome project (Adams et al., Science 287 (2000) 2185), and there are notable contradictions between the two models. The promoter and the 5' end of the transcript sequence were not identified in either model. In this paper, we present the gene structure of Dfos and identify the promoter. This promoter has an initiator and a downstream promoter element sequence, but lacks a TATA box. Through comparison of the mRNA and genomic DNA sequences, three introns varying in length from 66 bp to 17.57 kb were found and verified by RT-PCR. The Dfos gene is 21.2 kb in length, giving a transcript of 3438 bp, coding for a predicted protein of 595 amino acids. The 3' untranslated region is confirmed to be 1092 bp in length.