Atg9 is required for intraluminal vesicles in amphisomes and autolysosomes.

Autophagy is an intracellular recycling and degradation process, which is important for energy metabolism, lipid metabolism, physiological stress response and organism development. During Drosophila development, autophagy is up-regulated in fat body and midgut cells, to control metabolic function and to enable tissue remodelling. Atg9 is the only transmembrane protein involved in the core autophagy machinery and is thought to have a role in autophagosome formation. During Drosophila development, Atg9 co-located with Atg8 autophagosomes, Rab11 endosomes and Lamp1 endosomes-lysosomes. RNAi silencing of Atg9 reduced both the number and the size of autophagosomes during development and caused morphological changes to amphisomes/autolysosomes. In control cells there was compartmentalised acidification corresponding to intraluminal Rab11/Lamp-1 vesicles, but in Atg9 depleted cells there were no intraluminal vesicles and the acidification was not compartmentalised. We concluded that Atg9 is required to form intraluminal vesicles and for localised acidification within amphisomes/autolysosomes, and consequently when depleted, reduced the capacity to degrade and remodel gut tissue during development.

Identification and characterization of the STIM (stromal interaction molecule) gene family: coding for a novel class of transmembrane proteins.

STIM1 (where STIM is stromal interaction molecule) is a candidate tumour suppressor gene that maps to human chromosome 11p15.5, a region implicated in a variety of cancers, particularly embryonal rhabdomyosarcoma. STIM1 codes for a transmembrane phosphoprotein whose structure is unrelated to that of any other known proteins. The precise pathway by which STIM1 regulates cell growth is not known. In the present study we screened gene databases for STIM1-related sequences, and have identified and characterized cDNA sequences representing a single gene in humans and other vertebrates, which we have called STIM2. We identified a single STIM homologue in Drosophila melanogaster (D-Stim) and Caenorhabditis elegans, but no homologues in yeast. STIM1, STIM2 and D-Stim have a conserved genomic organization, indicating that the vertebrate family of two STIM genes most probably arose from a single ancestral gene. The three STIM proteins each contain a single SAM (sterile alpha-motif) domain and an unpaired EF hand within the highly conserved extracellular region, and have coiled-coil domains that are conserved in structure and position within the cytoplasmic region. However, the STIM proteins diverge significantly within the C-terminal half of the cytoplasmic domain. Differential levels of phosphorylation appear to account for two molecular mass isoforms (105 and 115 kDa) of STIM2. We demonstrate by mutation analysis and protein sequencing that human STIM2 initiates translation exclusively from a non-AUG start site in vivo. STIM2 is expressed ubiquitously in cell lines, and co-precipitates with STIM1 from cell lysates. This association into oligomers in vivo indicates a possible functional interaction between STIM1 and STIM2. The structural similarities between STIM1, STIM2 and D-STIM suggest conserved biological functions.

Receptor tyrosine kinase signaling regulates different modes of Groucho-dependent control of Dorsal.

Transcriptional control of the Drosophila terminal gap gene huckebein (hkb) depends on Torso (Tor) receptor tyrosine kinase (RTK) signaling and the Rel/NFkappaB homolog Dorsal (DI). DI acts as an intrinsic transcriptional activator in the ventral region of the embryo, but under certain conditions, such as when it is associated with the non-DNA-binding co-repressor Groucho (Gro), it is converted into a repressor. Gro is recruited to the enhancer element in the vicinity of DI by sequence-specific transcription factors such as Dead Ringer (Dri). We examined the interplay between DI, Gro and Dri on the hkb enhancer and show that when acting over a distance, Gro abolishes rather than converts DI activator function. Reducing the distance between DI- and Dri-binding sites, however, switches DI into a Gro-dependent repressor that overrides activation of transcription. Both of the distance-dependent regulatory options of Gro - quenching and silencing of transcription - are inhibited by RTK signaling. These data describe a newly identified mode of function for Gro when acting in concert with DI. RTK signaling provides a way of modulating DI function by interfering either with Gro activity or with Dri-dependent recruitment of Gro to the enhancer.

The Drosophila dead ringer gene is required for early embryonic patterning through regulation of argos and buttonhead expression.

The dead ringer (dri) gene of Drosophila melanogaster is a member of the recently discovered ARID-box family of eukaryotic genes that encode proteins with a conserved DNA binding domain. dri itself is highly conserved, with specific orthologs in the human, mouse, zebrafish and C. elegans genomes. We have generated dri mutant alleles to show that dri is essential for anterior-posterior patterning and for muscle development in the embryo. Consistent with the mutant phenotype and the sequence-specific DNA-binding properties of its product, dri was found to be essential for the normal early embryonic expression pattern of several key regulatory genes. In dri mutant embryos, expression of argos in the terminal domains was severely reduced, accounting for the dri mutant head phenotype. Conversely, buttonhead expression was found to be deregulated in the trunk region, accounting for the appearance of ectopic cephalic furrows. Curiously, dri was found also to be required for maintenance of expression of the ventrolateral region of even-skipped stripe four. This study establishes dri as an essential co-factor in the regulated expression of specific patterning genes during early embryogenesis.

Dorsal-mediated repression requires the formation of a multiprotein repression complex at the ventral silencer.

Dorsal functions as both an activator and repressor of transcription to determine dorsoventral fate in the Drosophila melanogaster embryo. Repression by Dorsal requires the corepressor Groucho (Gro) and is mediated by silencers termed ventral repression regions (VRRs). A VRR in zerknüllt (zen) contains Dorsal binding sites as well as an essential element termed AT2. We have identified and purified an AT2 DNA binding activity in embryos and shown it to consist of cut (ct) and dead ringer (dri) gene products. Studies of loss-of-function mutations in ct and dri demonstrate that both genes are required for the activity of the AT2 site. Dorsal and Dri both bind Gro, acting cooperatively to recruit it to the DNA. Thus, ventral repression may require the formation of a multiprotein complex at the VRR. This complex includes Dorsal, Gro, and additional DNA binding proteins, which appear to convert Dorsal from an activator to a repressor by enabling it to recruit Gro to the template. By showing how binding site context can dramatically alter transcription factor function, these findings help clarify the mechanisms responsible for the regulatory specificity of transcription factors.

[The mutagenic effect of plasmid DNA pUC19 and its derivatives]. / Mutagennyi éffekt plazmidnykh DNK pUC19 i ee proizvodnykh.

For a better understanding of the mechanism of foreign DNA mutagenesis, it is essential to study the validity of the changing DNA structure for manifestation of its mutagenic action on Drosophila melanogaster (method Basc). The testing of three different kinds of plasmid DNA has been carried out. The injections of only plasmid DNA pUC18 lead to a significant increase in the X-linked recessive lethal frequency. It is important to check mutagenicity of every new recombinant DNA.

[Transposition and reversion of mutations induced in Drosophila by viral DNA]. / Transpozitsii i reversii mutatsii drozofily, indutsirovannykh virusnoi DNK.

Injection of solutions of highly polymerized DNA isolated from nuclear polyhedrosis virus of Galleria mellonella into adult males induced with a considerable frequency visible mutations, two of which were studied in detail. They were detected in about 30 000 flies in the progeny of treated males. Much less than Notched-wings much greater than (Ndw, chromosome 3, location 87.9, dominant) independently arose 12 times, much less than thickened-veins much greater than (thi, chromosome 2, location 71.4, recessive) independently arose 7 times. No mutations were detected in the control of the same size. It was found that both Ndw and thi mutations gave frequent transpositions and reversions in mature, immature germ cells and in somatic cells, in latter cases leading to mosaicism. These results demonstrate for the first time that mutations induced by exogenous DNA are capable of transposition and reversion.