Drosophila neuroblasts act as stem cells. Their proliferation is controlled through cell cycle arrest and activation in a spatiotemporal pattern. Several genes have been identified that control the pattern of neuroblast quiescence and proliferation in the central nervous system (CNS), including anachronism (ana), even skipped (eve) and terribly reduced optic lobes (trol). eve acts in a non-cell-autonomous manner to produce a transacting factor in the larval body that stimulates cell division in the population of quiescent optic lobe neuroblasts. ana encodes a secreted glial glycoprotein proposed to repress premature proliferation of optic lobe and thoracic neuroblasts. trol was shown to act downstream of ana to activate proliferation of quiescent neuroblasts either by inactivating or bypassing ana-dependent repression. Here, we show that trol codes for Drosophila Perlecan, a large multidomain heparan sulfate proteoglycan originally identified in extracellular matrix structures of mammals. The results suggest that trol acts in the extracellular matrix and binds, stores, and sequesters external signals and, thereby, participates in the stage-and regionspecific control of neuroblast proliferation.

In both mammalian and insect models of ethanol-induced behavior, low doses of ethanol stimulate locomotion. However, the mechanisms of the stimulant effects of ethanol on the CNS are mostly unknown. We have identified tao, encoding a serine-threonine kinase of the Ste20 family, as a gene necessary for ethanol-induced locomotor hyperactivity in Drosophila. Mutations in tao also affect behavioral responses to cocaine and nicotine, making flies resistant to the effects of both drugs. We show that tao function is required during the development of the adult nervous system and that tao mutations cause defects in the development of central brain structures, including the mushroom body. Silencing of a subset of mushroom body neurons is sufficient to reduce ethanol-induced hyperactivity, revealing the mushroom body as an important locus mediating the stimulant effects of ethanol. We also show that mutations in par-1 suppress both the mushroom body morphology and behavioral phenotypes of tao mutations and that the phosphorylation state of the microtubule-binding protein Tau can be altered by RNA interference knockdown of tao, suggesting that tao and par-1 act in a pathway to control microtubule dynamics during neural development.

Alcelaphine herpesvirus 1 (AHV-1) causes wildebeest-associated malignant catarrhal fever, a lymphoproliferative syndrome in ungulate species other than the natural host. Based on biological properties and limited structural data, it has been classified as a member of the genus Rhadinovirus of the subfamily Gammaherpesvirinae. Here, we report on cloning and structural analysis of the complete genome of AHV-1 C500. The low GC content DNA (L-DNA) region of the genome consists of 130,608 bp with low (46.17%) GC content and marked suppression of CpG dinucleotide frequency. Like in herpesvirus saimiri, the prototype of the rhadinoviruses, the L-DNA is flanked by approximately 20 to 25 GC-rich (71.83%) high GC content DNA (H-DNA) repeats of 1,113 to 1,118 nucleotides. The analysis of the L-DNA sequence revealed 70 open reading frames (ORFs), 61 of which showed homology to other herpesviruses. The conserved ORFs are arranged in four blocks collinear to other Rhadinovirus genomes. These gene blocks are flanked by nonconserved regions containing ORFs without similarities to known herpesvirus genes. Notably, a spliced reading frame with a coding capacity for a 199-amino-acid protein is located in a position homologous to the transforming genes of herpesvirus saimiri at the left end of the L-DNA. A gene with homology to the semaphorin family is located adjacent to this. Despite common biological and epidemiological properties, AHV-1 differs significantly from herpesvirus saimiri with regard to cell homologous genes, probably using a different set of effector proteins to achieve a similar T-lymphocyte-transforming phenotype. MATERIALS AND METHODS Cell lines and viral culture. The bovine epithelial kidney cell line MDBK (ATCC CCL 22) was propagated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 g of gentamicin per ml, and 350 g of L-glutamine per ml. AHV-1 strains C500 (62) and WC11 (63) were serially propagated by infection of fresh MDBK cells with aliquots of infected MDBK cells showing cytopathic changes in more than 80% of the cells. AHV-1 C500 was obtained from H. W. Reid (Moredun Research Institute, Edinburgh, United Kingdom), and the attenuated WC11 strain was obtained from D. W. Verwoerd (Veterinary Research Institute, Onderstepoort, South Africa). For the preparation of virions, tissue culture supernatant was precleared by centrifugation at 2,000 ϫ g. Strain C500 virions (passage 5) were pelleted by centrifugation at 50,000 ϫ g with an SW28 rotor. The viral DNA was extracted from the pellet with phenol-chloroform and precipitated with ethanol. WC11 DNA was further purified by density gradient centrifugation as described previously (37). Cloning procedures.

The transition from a liquid to a gas filled tubular network is the prerequisite for normal function of vertebrate lungs and invertebrate tracheal systems. However, the mechanisms underlying the process of gas filling remain obscure. Here we show that waterproof, encoding a fatty acyl-CoA reductase (FAR), is essential for the gas filling of the tracheal tubes during Drosophila embryogenesis, and does not affect branch network formation or key tracheal maturation processes. However, electron microscopic analysis reveals that in waterproof mutant embryos the formation of the outermost tracheal cuticle sublayer, the envelope, is disrupted and the hydrophobic tracheal coating is damaged. Genetic and gain-of-function experiments indicate a non-cell-autonomous waterproof function for the beginning of the tracheal gas filling process. Interestingly, Waterproof reduces very long chain fatty acids of 24 and 26 carbon atoms to fatty alcohols. Thus, we propose that Waterproof plays a key role in tracheal gas filling by providing very long chain fatty alcohols that serve as potential substrates for wax ester synthesis or related hydrophobic substances that ultimately coat the inner lining of the trachea. The hydrophobicity in turn reduces the tensile strength of the liquid inside the trachea, leading to the formation of a gas bubble, the focal point for subsequent gas filling. Waterproof represents the first enzyme described to date that is necessary for tracheal gas filling without affecting branch morphology. Considering its conservation throughout evolution, Waterproof orthologues may play a similar role in the vertebrate lung.

Protein cross-linking and the analysis of cross-linked peptides by mass spectrometry is currently receiving much attention. Not only is this approach applied to isolated complexes to provide information about spatial arrangements of proteins, but it is also increasingly applied to entire cells and their organelles. As in quantitative proteomics, the application of isotopic labeling further makes it possible to monitor quantitative changes in the protein-protein interactions between different states of a system. Here, we cross-linked mitochondria from Saccharomyces cerevisiae grown on either glycerol- or glucose-containing medium to monitor protein-protein interactions under non-fermentative and fermentative conditions. We investigated qualitatively the protein-protein interactions of the 400 most abundant proteins applying stringent data-filtering criteria, i.e. a minimum of two cross-linked peptide spectrum matches and a cut-off in the spectrum scoring of the used search engine. The cross-linker BS3 proved to be equally suited for connecting proteins in all compartments of mitochondria when compared with its water-insoluble but membrane-permeable derivative DSS. We also applied quantitative cross-linking to mitochondria of both the growth conditions using stable-isotope labeled BS3. Significant differences of cross-linked proteins under glycerol and glucose conditions were detected, however, mainly because of the different copy numbers of these proteins in mitochondria under both the conditions. Results obtained from the glycerol condition indicate that the internal NADH:ubiquinone oxidoreductase Ndi1 is part of an electron transport chain supercomplex. We have also detected several hitherto uncharacterized proteins and identified their interaction partners. Among those, Min8 was found to be associated with cytochrome c oxidase. BN-PAGE analyses of min8Δ mitochondria suggest that Min8 promotes the incorporation of Cox12 into cytochrome c oxidase.