A humoral stress response in Drosophila.

The ability to react to unfavorable environmental changes is crucial for survival and reproduction, and several adaptive responses to stress have been conserved during evolution [1-3]. Specific immune and heat shock responses mediate the elimination of invading pathogens and of damaged proteins or cells [4-6]. Furthermore, MAP kinases and other signaling factors mediate cellular responses to a very broad range of environmental insults [7-9]. Here we describe a novel systemic response to stress in Drosophila. The Turandot A (TotA) gene encodes a humoral factor, which is secreted from the fat body and accumulates in the body fluids. TotA is strongly induced upon bacterial challenge, as well as by other types of stress such as high temperature, mechanical pressure, dehydration, UV irradiation, and oxidative agents. It is also upregulated during metamorphosis and at high age. Strikingly, flies that overexpress TotA show prolonged survival and retain normal activity at otherwise lethal temperatures. Although TotA is only induced by severe stress, it responds to a much wider range of stimuli than heat shock genes such as hsp70 or immune genes such as Cecropin A1.

Two attacin antibacterial genes of Drosophila melanogaster.

Insects express a battery of potent antimicrobial proteins in response to injury and infection. Recent work from several laboratories has demonstrated that this response is neither stereotypic nor completely nonspecific, and that different pathways are responsible for inducing the expression of antifungal and antibacterial peptides. Here we report the cloning of two closely linked attacin genes from Drosophila melanogaster. We compare their protein coding sequences and find the amino acid sequences to be more highly conserved than the nucleotide sequences, suggesting that both genes are expressed. Like other antimicrobial peptides, attacin expression is strongly induced in infected and injured flies. Unlike others, attacin transcription is uniquely sensitive to mutations in the 18-Wheeler receptor protein, and thus may be regulated by a distinct signaling pathway. The number and organization of binding sites for kappaB and other transcription factors in the promoter regions of both attacin genes are consistent with strong and rapid immune induction. We demonstrate that these promoter regions are sufficient to direct beta-galactosidase expression in transformed Drosophila third-instar larval fat body in a bacterially inducible manner. We present a comparison of the promoter regions of the two attacin genes to those cloned from other antimicrobial peptide genes to assist a better understanding of how antimicrobial genes are differentially regulated.

Relish, a central factor in the control of humoral but not cellular immunity in Drosophila.

The NF-kappa B-like Relish gene is complex, with four transcripts that are all located within an intron of the Nmdmc gene. Using deletion mutants, we show that Relish is specifically required for the induction of the humoral immune response, including both antibacterial and antifungal peptides. As a result, the Relish mutants are very sensitive to infection. A single cell of E. cloacae is sufficient to kill a mutant fly, and the mutants show increased susceptibility to fungal infection. In contrast, the blood cell population, the hematopoietic organs, and the phagocytic, encapsulation, and melanization responses are normal. Our results illustrate the importance of the humoral response in Drosophila immunity and demonstrate that Relish plays a key role in this response.

Origins of immunity: Relish, a compound Rel-like gene in the antibacterial defense of Drosophila.

NF-kappa B/Rel transcription factors are central regulators of mammalian immunity and are also implicated in the induction of cecropins and other antibacterial peptides in insects. We identified the gene for Relish, a compound Drosophila protein that, like mammalian p105 and p100, contains both a Rel homology domain and an I kappa B-like domain. Relish is strongly induced in infected flies, and it can activate transcription from the Cecropin A1 promoter. A Relish transcript is also detected in early embryos, suggesting that it acts in both immunity and embryogenesis. The presence of a compound Rel protein in Drosophila indicates that similar proteins were likely present in primordial immune systems and may serve unique signaling functions.

Identification of early genes in the Drosophila immune response by PCR-based differential display: the Attacin A gene and the evolution of attacin-like proteins.

We are using the PCR-based differential display technique to isolate genes which are induced during the immune response in Drosophila. In this way, a cDNA clone for a member of the attacin family of antibacterial proteins was isolated. The corresponding Attacin A (Att A) gene is localized at 51A-B on the second chromosome, and it is closely linked to at least one more cross-hybridizing gene. Injection of bacteria induces a 0.8 kb transcript, with expression kinetics similar to that of cecropin. Drosophila attacin is most closely related to sarcotoxin II of Sarcophaga peregrina, but it lacks the extra domains that are unique to this protein, and the overall domain structure of the Att A gene product is identical to that of the attacins from Hyalophora cecropia.

Behavior in light-dark cycles of Drosophila mutants that are arrhythmic, blind, or both.

Certain of the rhythm mutations in Drosophila melanogaster lead to arrhythmic locomotor activity (and aperiodic eclosion) in constant conditions. In light-dark (LD) cycles, however, such mutants exhibit clear fluctuations between high levels of activity when the lights are on and much lower ones when they are off. Our data, in contrast to some previous conclusions, strongly suggest that period0 (per0) adults are, in LD conditions, merely being "forced" into exhibiting periodic behavior. These experiments involved application of 8-, 12-, 16-, and 24-hr LD cycles, in which the arrhythmic mutant could have any of these periodicities imposed upon it, whereas wild-type flies tended to exhibit periods of about 24 hr in cycling conditions whose T values were > 8 hr different from 24. In phase-shift experiments, it was found that Drosophila expressing genotypes associated with rhythmicity achieved a 5-hr advance over a 2-day period following an advanced lights-on; per0 adults altered the phase of their locomotor peaks more rapidly. Against a background of the fact that eyeless or blind flies exhibit normal entrainment, it was hypothesized that double-mutant flies--carrying such visual mutations and per0 as well--should not synchronize to LD cycles, if the forced rhythms seen in the latter single-mutant type are mediated solely by light input through the external photoreceptors. Since an appreciable proportion of the double mutants did synchronize (to LD 12:12), it is thus suggested that the visual cues involved in forcing rhythmicity could be input through the same extraocular photoreceptors that, in general, subserve the fly's rhythm system.

Mapping the clock rhythm mutation to the period locus of Drosophila melanogaster by germline transformation.

The Clock (Clk) mutation shortens circadian rhythms of locomotor activity and eclosion from ca. 24 h to 22.5-23 h. Clk was previously mapped, by meiotic recombination, very close to the period(per) locus on the X chromosome. To determine whether Clk is a mutation within the per gene or if the former is separate from the latter, two overlapping genomic fragments were cloned from Clk flies to produce a per-containing 13.2 kb construct, per01 flies (which by themselves are arrhythmic)--when transformed with this construct--expressed short-period rhythms. This indicates that the Clk mutation is contained within this 13.2 kb region and is almost certainly a new "fast-clock" allele of per.

Phenotypic and genetic analysis of Clock, a new circadian rhythm mutant in Drosophila melanogaster.

Clock is a semidominant X-linked mutation that results in shortening the period of Drosophila melanogaster's free-running locomotor activity rhythm from ca. 24.0 to ca. 22.5 hr. This mutation similarly shortened the phase response curve, determined by resetting activity rhythms with light pulses. Eclosion peaks for Clk cultures were separated by only 22.5 hr instead of the normal 24 hr. Clk was mapped close to, but separable from, another rhythm mutation--period01--by recombination. The estimated distance between these two mutations was short enough to suggest that Clk could be a per allele. If this is the case, the new mutant is unique in that it, unlike other per variants, is associated with essentially normal 1-min courtship song rhythms when Clk is expressed in males. Also, the new rhythm variant could not, in contrast to a short-period per mutation, have its effects on free-running activity rhythms uncovered by deletions. This result, and the lack of coverage of Clk's effects by duplications, suggest that it is not a simple hypomorphic or amorphic mutation.

High-resolution analysis of locomotor activity rhythms in disconnected, a visual-system mutant of Drosophila melanogaster.

Free-running locomotor activity and eclosion rhythms of Drosophila melanogaster, mutant at the disconnected (disco) locus, are substantially different from the wild-type phenotype. Initial periodogram analysis revealed little or no rhythmicity (Dushay et al., 1989). We have reanalyzed the locomotor activity data using high-resolution signal analysis (maximum-entropy spectral analysis, or MESA). These analyses, corroborated by autocorrelograms, uncovered significant residual circadian rhythmicity and strong ultradian rhythms in most of the animals tested. In this regard the disco mutants are much like flies expressing mutant alleles of the period gene, as well as wild-type flies reared throughout life in constant darkness. We hypothesize that light normally triggers the coupling of multiple ultradian oscillators into a functional circadian clock and that this process is disrupted in disco flies as a result of the neural lesion.

The disconnected visual system mutations in Drosophila melanogaster drastically disrupt circadian rhythms.

Mutations at the disconnected (disco) locus in Drosophila melanogaster cause cultures of this insect to eclose in an essentially arrhythmic manner and also nearly eliminate free-running circadian rhythms of locomotor activity. Yet disco mutants are not totally light-insensitive: Whereas they performed very poorly in tests of certain behavioral responses to visual stimuli, they were able to exhibit "forced" periodic locomotor activity under conditions of light-dark cycling. We discuss these results in the context of (1) the dispensability of this insect's external photoreceptors for entrainment of its circadian pacemaker, and (2) possible disco-induced abnormalities in the connections of extraocular photoreceptors to their targets in the central nervous system and/or abnormalities in the targets themselves--which presumably include elements of the fly's circadian clock.