Role and activation of type III secretion system genes in Pseudomonas aeruginosa-induced Drosophila killing.

Pseudomonas aeruginosa strains PAO1 and CHA showing type III system-dependent cytotoxicity towards macrophages ex vivo are able to induce rapid death of adult fly Drosophila melanogaster accompanied by bacterial multiplication to high-titers. The role of P. aeruginosa type III secretion system in rapid fly killing was demonstrated here by using several isogenic CHA mutants, selectively affected in this system. The activation of P. aeruginosa pexsCBA, the regulatory operon of the type III system, and the activation of the Drosophila gene diptericin, showed the host-pathogen recognition during infection process.

Dominant modifiers of the polyhomeotic extra-sex-combs phenotype induced by marked P element insertional mutagenesis in Drosophila.

Members of the Polycomb group (Pc-G) and trithorax group (trx-G) of genes, as well as the enhancers of trx-G and Pc-G (ETP), function together to maintain segment identity during Drosophila development. In order to obtain new marked P mutations in these genes, we screened for dominant modifiers of the extra-sex-combs phenotype displayed by males mutant for the polyhomeotic (ph) gene, a member of the Pc-G group. Five P(lacW) insertions in four different genes were found to stably suppress ph: two are allelic to trithorax, one is the first allele specific to the Minute(2)21C gene, and the remaining two define new trx-G genes, toutatis (tou) in 48A and taranis (tara) in 89B10-13. tou is predicted to encode a 3109 amino acid sequence protein (TOU), which contains a TAM DNA-binding domain, a WAKZ motif, two PHD zinc fingers and a C-terminal bromodomain, and as such is likely to be involved in regulation of chromatin structure as a subunit of a novel chromatin remodelling complex. In a previous study, we found that insertion of a P(ph) transposable element containing ph regulatory sequences creates a high frequency of mutations modifying ph homeotic phenotypes. One such insertion enhanced the ph phenotype and we show that it is a new allele of UbcD1/eff, a gene encoding a ubiquitin-conjugating enzyme that is involved in telomere association and potentially in chromatin remodelling.

RotundRacGAP functions with Ras during spermatogenesis and retinal differentiation in Drosophila melanogaster.

Our analysis of rotund (rn) null mutations in Drosophila melanogaster revealed that deletion of the rn locus affects both spermatid and retinal differentiation. In the male reproductive system, the absence of RnRacGAP induced small testes, empty seminal vesicles, short testicular cysts, reduced amounts of interspermatid membrane, the absence of individualization complexes, and incomplete mitochondrial condensation. Flagellar growth continued within the short rn null cysts to produce large bulbous terminations of intertwined mature flagella. Organization of the retina was also severely perturbed as evidenced by grossly misshapen ommatidia containing reduced numbers of photoreceptor and pigment cells. These morphological phenotypes were rescued by genomic rnRacGAP transgenes, demonstrating that RnRacGAP function is critical to spermatid and retinal differentiation. The testicular phenotypes were suppressed by heterozygous hypomorphic mutations in the Dras1 and drk genes, indicating cross talk between RacGAP-regulated signaling and that of the Ras pathway. The observed genetic interactions are consistent with a model in which Rac signaling is activated by Ras and negatively regulated by RnRacGAP during spermatid differentiation. RnRacGAP and Ras cross talk also operated during retinal differentiation; however, while the heterozygous hypomorphic drk mutation continued to act as a suppressor of the rn null mutation, the heterozygous hypomorphic Dras1 mutation induced novel retinal phenotypes.

The Rac GTPase-activating protein RotundRacGAP interferes with Drac1 and Dcdc42 signalling in Drosophila melanogaster.

RhoGTPases are negatively regulated by GTPase-activating proteins (GAPs). Here we demonstrate that Drosophila RotundRacGAP is active in vitro on Drac1 and Dcdc42 but not Drho1. Similarly, in yeast, RotundRacGAP interacts specifically with Drac1 and Dcdc42, as well as with their activated V12 forms, showing a particularly strong interaction with Dcdc42V12. In the fly, lowering RotundRacGAP dosage specifically modifies eye defects induced by expressing Drac1 or Dcdc42 but not Drho1, confirming that Drac1 and Dcdc42 are indeed in vivo targets of RotundRacGAP. Furthermore, embryonic-directed expression of either RotundRacGAP, or dominant negative Drac1N17, transgenes induces similar defects in dorsal closure and inhibits Drac1-dependent cytoskeleton assembly at the leading edge. Expression of truncated forms of RotundRacGAP shows that the GAP domain of RotundRacGAP is essential for its function. Unexpectedly, transgenes encoding Drac1N17, Dcdc42N17, or RotundRacGAP do not affect the c-Jun N-terminal kinase-dependent gene expression of decapentaplegic and puckered, indicating that another Drac1-independent signal redundantly activates this pathway. Finally, in a situation where Drac1 is constitutively activated, RotundRacGAP greatly reduces the ectopic expression of decapentaplegic, possibly by negatively regulating Dcdc42.

One-to-one correspondence between the two genetic units and the tandemly duplicated transcriptional units of the polyhomeotic locus of Drosophila.

polyhomeotic (ph) is a complex locus in Drosophila defined by two genetic units. Two mutational events are necessary to obtain the null lethal phenotype. Molecular analysis has shown that the ph locus contains two transcriptional units coding for two very similar proteins. Although a strong argument in favor of a strict correlation between the genetic and molecular units can be constructed, there is no direct evidence for the hypothesis. Here, we show for all cases with detectable molecular defects that X-ray-induced generation of an amorphic allele from a pre-existing X-ray-induced hypomorphic allele with a lesion limited to one unit invariably involves a rearrangement in the other unit. This result proves that each genetic unit corresponds to one transcription unit.

white+ transgene insertions presenting a dorsal/ventral pattern define a single cluster of homeobox genes that is silenced by the polycomb-group proteins in Drosophila melanogaster.

We used the white gene as an enhancer trap and reporter of chromatin structure. We collected white+ transgene insertions presenting a peculiar pigmentation pattern in the eye: white expression is restricted to the dorsal half of the eye, with a clear-cut dorsal/ventral (D/V) border. This D/V pattern is stable and heritable, indicating that phenotypic expression of the white reporter reflects positional information in the developing eye. Localization of these transgenes led us to identify a unique genomic region encompassing 140 kb in 69D1-3 subject to this D/V effect. This region contains at least three closely related homeobox-containing genes that are constituents of the iroquois complex (IRO-C). IRO-C genes are coordinately regulated and implicated in similar developmental processes. Expression of these genes in the eye is regulated by the products of the Polycomb-group (Pc-G) and trithorax-group (trx-G) genes but is not modified by classical modifiers of position-effect variegation. Our results, together with the report of a Pc-G binding site in 69D, suggest that we have identified a novel cluster of target genes for the Pc-G and trx-G products. We thus propose that ventral silencing of the whole IRO-C in the eye occurs at the level of chromatin structure in a manner similar to that of the homeotic gene complexes, perhaps by local compaction of the region into a heterochromatin-like structure involving the Pc-G products.

Regulation of polyhomeotic transcription may involve local changes in chromatin activity in Drosophila.

The polyhomeotic (ph) gene of Drosophila is a member of the Polycomb group of genes and encodes a chromatin protein required for negative regulation of homeotic genes and other loci, in particular the ph locus itself. We have studied the genetic control of ph transcription during development. Early ph expression is under the control of bicoid and engrailed as activators and of oskar as an inhibitor. The negative autoregulation of ph starts at the blastoderm stage and is partly mediated by a transvection effect. As the number of functional copies of ph increases in the same genome, a concomitant reduction of the transcription of each copy is observed. This regulation is ensured, likely at the chromatin level, positively by the trithorax group and negatively by the Polycomb group gene products like a homeotic gene, but it occurs in the same cells. We propose that an equilibrium between these two states of chromatin activity ensures an accurate level of ph transcription.

polyhomeotic regulatory sequences induce developmental regulator-dependent variegation and targeted P-element insertions in Drosophila.

Variegation of the miniwhite gene is observed in a euchromatic context in transformant lines that contain a P transposon including regulatory sequences of the polyhomeotic (ph) gene upstream of the resident miniwhite gene (P[ph]). This variegated phenotype is not affected by most of the genetic modifiers of heterochromatic position-effect variegation (PEV) nor by removal of the Y chromosome. Interestingly, it is sensitive to ph and Polycomb (Pc) mutations, which are known to affect homeotic gene regulation. Regulatory DNA of ph can also mediate transvection of the miniwhite gene. This transvection is abolished in a ph but not in a zeste mutant background. In addition, P[ph] inserts preferentially in sites corresponding to PH/PC protein-binding sites as defined at the polytene chromosome level. These insertions induce an unusually high proportion of mutations in genes affecting homeotic gene regulation. In particular, one insertion is located within the tramtrack locus, which is thought to regulate fushi tarazu, an Ultrabithorax activator. We suggest that a multimeric complex containing PH and PC proteins, at a minimum, causes a local and clonally inherited heterochromatinization, which maintains the repressed state of transcription of the homeotic genes.

A Brassica napus transcript encoding a protein related to the Künitz protease inhibitor family accumulates upon water stress in leaves, not in seeds.

A cDNA clone encoding a Brassica napus drought-induced 22 kDa (BnD22) protein has been isolated and characterized. The BnD22 transcript accumulated in response to drought reversibly, and to other conditions of leaf water deficit such as rapid water stress or salt acclimation, but not to cold acclimation or heat shock. Exogenously applied abscisic acid induced both changes in leaf morphology similar to the drought-adaptive response and a pronounced accumulation of the BnD22 mRNA. In control and drought-adapted plants, the BnD22 transcript was expressed in an organ-specific manner: the mRNA level was highest in leaves, low in hypocotyls and undetectable in roots. Sequence analysis indicates that the BnD22 protein is related to the Künitz family of protease inhibitors. In contrast to most members of this family, and also to most polypeptides expressed in vegetative tissues upon drought, the BnD22 mRNA was absent in seeds, before or during the seed desiccation phase. The BnD22 gene represents a new class of genes which are strictly induced in vegetative tissues upon environmental stress, and its pattern of expression shows that the responses to water deficit differ, at least partially, in seeds and in leaves.

The unusual reversion properties of a mitochondrial mutation in the structural gene of subunit I of cytochrome oxidase of Saccharomyces cerevisiae reveal a probable histidine ligand of the redox center.

We have analyzed a mutation in the mitochondrial gene oxi3 coding for subunit I of cytochrome-oxidase in the yeast Saccharomyces cerevisiae. This mutation replaces one of the seven invariant histidines of the polypeptide (position 378) by a tyrosine, and leads to a respiratory deficient phenotype. A total of 157 revertants, which have recovered the ability to grow on a respiratory substrate, have been selected from this mutant (tyrosine 378). The nature of the reversion has been analysed by a rapid screening procedure and 32 of the revertants have been sequenced. They are all true back-mutations reintroducing the histidine in position 378. This very exceptional situation suggests that this histidine is a ligand of the redox center of cytochrome oxidase.