Spermidine promotes stress resistance in Drosophila melanogaster through autophagy-dependent and -independent pathways

The naturally occurring polyamine spermidine (Spd) has recently been shown to promote longevity across species in an autophagy-dependent manner. Here, we demonstrate that Spd improves both survival and locomotor activity of the fruit fly Drosophila melanogaster upon exposure to the superoxide generator and neurotoxic agent paraquat. Although survival to a high paraquat concentration (20 mM) was specifically increased in female flies only, locomotor activity and survival could be rescued in both male and female animals when exposed to lower paraquat levels (5 mM). These effects are dependent on the autophagic machinery, as Spd failed to confer resistance to paraquat-induced toxicity and locomotor impairment in flies deleted for the essential autophagic regulator ATG7 (autophagy-related gene 7). Spd treatment did also protect against mild doses of another oxidative stressor, hydrogen peroxide, but in this case in an autophagy-independent manner. Altogether, this study establishes that the protective effects of Spd can be exerted through different pathways that depending on the oxidative stress scenario do or do not involve autophagy.     

COM,a heterochromatic locus governing the control of independent endogenous retroviruses from Drosophila melanogaster

ZAM and Idefix are two endogenous retroviruses whose expression is tightly controlled in Drosophila melanogaster. However, a line exists in which this control has been perturbed, resulting in a high mobilization rate for both retroviruses. This line is called the U (unstable) line as opposed to the other S (stable) lines. In the process of analyzing this control and tracing the genetic determinant involved, we found that ZAM and Idefix expression responded to two types of controls: one restricting their expression to specific somatic cells in the ovaries and the other silencing their expression in S lines but permitting it in U lines. While studying this second control in the U or S backgrounds, we found that the heterochromatic locus 20A2-3 on the X chromosome, previously implicated in the regulation of a third retroelement, gypsy, also controlled both ZAM and Idefix. We report here that genetic determinants necessary for endogenous retrovirus silencing occur at the 20A2-3 locus, which we call COM, for centre organisateur de mobilisation. We propose that if this point of control becomes mutated during the life of the fly, it may trigger processes reactivating dormant endogenous retroviruses and thus bring about sudden bursts of mobilization.     

Genotoxic effects of two nickel-compounds in somatic cells of Drosophila melanogaster

In view of the scarcely available information on the in vivo mutagenic and co-mutagenic activity of nickel, the genotoxic potential of two nickel-compounds, nickel chloride (NiCl(2)) and nickel sulphate (NiSO(4)), was assessed in Drosophila melanogaster by measuring two different genetic endpoints. On the one hand, we used the wing-spot assay, which is based on the principle that the loss of heterozygosity of two suitable recessive markers, multiple wing hairs (mwh) and flare-3 (flr(3)), can lead to the formation of mutant clones in the imaginal disks of larval cells. On the other hand, the in vivo comet assay, which detects single- and double-strand DNA breaks, was also used with larval haemocytes. These cells offer several advantages: they are highly sensitive to genotoxic agents, the sampling and processing methodologies are quite simple and the level of basal DNA damage is relatively low. No significant increases in the frequencies of the three categories of mutant spots (i.e. small single spots, large single spots, and twin spots) were observed in the wing-spot assay; however, NiSO(4) induced significant dose-dependent increases in DNA damage in the comet assay. In addition, the combined treatments with gamma-radiation and NiCl(2) and NiSO(4) showed a slight but significant increase in the frequency of the three categories of mutant spots compared with the frequency induced by gamma-radiation alone, indicating that both nickel compounds have a synergistic interaction. These results support the assumption that both nickel compounds could act as co-mutagens interfering with DNA-repair processes and that the in vivo comet assay is a sensitive and effective method for detecting the DNA damage induced by NiSO(4) in haemocytes of D. melanogaster.     

MDG1het and aurora--non-mobile retrotransposons of Drosophila melanogaster]

Non-mobile retrotransposons mdg1het and aurora localized in Drosophila melanogaster heterochromatin were studied. A novel retrotransposon aurora comprising 324 bp LTRs was revealed as a 5 kb insertion causing 5 bp duplication of integration site in the heterochromatic Stellate gene. All the aurora copies are immobilized in D. melanogaster heterochromatin and adjoining chromosome regions 40, 41C and 80BC. Mobile aurora copies were revealed in D. simulans euchromatin by in situ hybridization technique. A comparison of 2.5 kb sequence of immobile mdg1het (including a half of ORF2 and 3'-LTR) with the correspondent sequence of transposable mdg1 copy [9] allowed to conclude that evolution of mdg1 subfamilies occurred under the selective pressure for the ability to transpose. The time period passed since the aurora and mdg1 copies integrated in heterochromatin was roughly estimated via divergence extent between the left and right LTR; for aurora copy it is 0-0.15 Myr, and for mdg1het copies it is 0-0.7 Myr.     

Drosophila PTEN regulates cell growth and proliferation through PI3K-dependent and -independent pathways

The control of cell and organ growth is fundamental to the development of multicellular organisms. Here, we show that dPTEN, a Drosophila homolog of the mammalian PTEN tumor suppressor gene, plays an essential role in the control of cell size, cell number, and organ size. In mosaic animals, dPTEN(-) cells proliferate faster than their heterozygous siblings, show an autonomous increase in cell size, and form organs of increased size, whereas overexpression of dPTEN results in opposite phenotypes. The loss-of-function phenotypes of dPTEN are suppressed by mutations in the PI3K target Dakt1 and the translational initiation factor eif4A, suggesting that dPTEN acts through the PI3K signaling pathway to regulate translation. Although activation of PI3K and Akt has been reported to increase rates of cellular growth but not proliferation, loss of dPTEN stimulates both of these processes, suggesting that PTEN regulates overall growth through PI3K/Akt-dependent and -independent pathways. Furthermore, we show that dPTEN does not play a major role in cell survival during Drosophila development. Our results provide a potential explanation for the high frequency of PTEN mutation in human cancer.     

Transposition of the bobbed locus in Drosophila melanogaster

Due to the complete absence of ribosomal DNA (genetic symbol bb-), the Xbb- chromosome of Drosophila is lethal both in homozygous conditions and in compound with the Xbb- chromosome. However, in the cross between the C(1)RM/Ybb- females and the Xbb-/BSYbb+ males, characterized by the development of lethal Xbb-/Ybb- zygotes, two fertile males were detected. These males possessed all the markers of the Xbb- chromosome but lacked the Y chromosome BS marker. Genetic analysis of their progeny showed that genes responsible for restoration of viability and fertility of these exceptional males were associated with the X chromosome. The crossover tests showed that in one case these genes were tightly linked to the w locus (the bbAM1 allele), and in the second case they were located 12.6 map units to the right of the Tu locus (the bbAM7 allele). It has also been shown that the bb locus was transposed to the X chromosome within the short arm of Y chromosome. Transposition of the BSYbb+ chromosome-specific rDNA sequences to the X chromosome was confirmed by means of Southern blotting. These data indicate that replacement of the bb locus is realized by transposition rather than recombination.     

In vivo phosphorylation of Drosophila melanogaster nuclear lamins during both interphase and mitosis

To study phosphorylation of D. melanogaster nuclear lamins in vivo, we used Kc tissue culture cells. Kc cells contain products of both lamin genes, the lamin Dm0 gene encoding constitutive polypeptides expressed in almost all cell types and the developmentally regulated lamin C gene. We grew Kc cells in low phosphate medium and labelled them with (32P(H3PO4. To obtain mitotic cells we used vinblastine to arrest cells in metaphase. Cells were collected, washed, lysed and resultant extracts fractionated in the presence of protein phosphatase inhibitors. D. melanogaster proteins were then denatured by boiling in SDS plus DTT, followed by immunoaffinity chromatography and SDS-PAGE purification. As anticipated, we found that a CNBr fragment derived from the N-terminal part of lamin Dm0-derivatives (amino acid residues 2-158; fragment A) was phosphorylated during both interphase and mitosis. Interphase but not mitotic phosphorylation was found on an internal CNBr fragment (derived from the end of the central rod domain and the first part of the C-terminal lamin tail; amino acid residues 385-548; fragment D). Interphase only phosphorylation was also detected on another CNBr fragment derived from the extreme C-terminal portion of lamin Dm0-derivatives (amino acid residues 549-622; fragment E). To supplement these data, we used 2-D tryptic peptide mapping followed by phosphorImager analysis. We routinely detected at least seven 'spots' derived from interphase lamins but only a single mitotic lamin phosphopeptide.     

Transvection at the vestigial locus of Drosophila melanogaster

Transvection is a phenomenon wherein gene expression is effected by the interaction of alleles in trans and often results in partial complementation between mutant alleles. Transvection is dependent upon somatic pairing between homologous chromosome regions and is a form of interallelic complementation that does not occur at the polypeptide level. In this study we demonstrated that transvection could occur at the vestigial (vg) locus by revealing that partial complementation between two vg mutant alleles could be disrupted by changing the genomic location of the alleles through chromosome rearrangement. If chromosome rearrangements affect transvection by disrupting somatic pairing, then combining chromosome rearrangements that restore somatic pairing should restore transvection. We were able to restore partial complementation in numerous rearrangement trans-heterozygotes, thus providing substantial evidence that the observed complementation at vg results from a transvection effect. Cytological analyses revealed this transvection effect to have a large proximal critical region, a feature common to other transvection effects. In the Drosophila interphase nucleus, paired chromosome arms are separated into distinct, nonoverlapping domains. We propose that if the relative position of each arm in the nucleus is determined by the centromere as a relic of chromosome positions after the last mitotic division, then a locus will be displaced to a different territory of the interphase nucleus relative to its nonrearranged homolog by any rearrangement that links that locus to a different centromere. This physical displacement in the nucleus hinders transvection by disrupting the somatic pairing of homologous chromosomes and gives rise to proximal critical regions.     

HeT-A telomere-specific retrotransposons in the centric heterochromatin of Drosophila melanogaster chromosome 3

We have isolated two yeast artificial chromosome (YAC) clones from Drosophila melanogaster that contain a small amount of dodeca satellite (a satellite DNA located in the centromeric region of chromosome 3) and sequences homologous to the telomeric retrotransposon HeT-A. Using these YACs as probes for fluorescence in situ hybridization to mitotic chromosomes, we have localized these HeT-A elements to the centric heterochromatin of chromosome 3, at region h55. The possible origin of these telomeric elements in a centromeric position is discussed. Received: 30 July 1999 / Accepted: 19 September 1999     

Transvection and silencing of the Scr homeotic gene of Drosophila melanogaster

The Sex combs reduced (Scr) gene specifies the identities of the labial and first thoracic segments in Drosophila melanogaster. In imaginal cells, some Scr mutations allow cis-regulatory elements on one chromosome to stimulate expression of the promoter on the homolog, a phenomenon that was named transvection by Ed Lewis in 1954. Transvection at the Scr gene is blocked by rearrangements that disrupt pairing, but is zeste independent. Silencing of the Scr gene in the second and third thoracic segments, which requires the Polycomb group proteins, is disrupted by most chromosomal aberrations within the Scr gene. Some chromosomal aberrations completely derepress Scr even in the presence of normal levels of all Polycomb group proteins. On the basis of the pattern of chromosomal aberrations that disrupt Scr gene silencing, we propose a model in which two cis-regulatory elements interact to stabilize silencing of any promoter or cis-regulatory element physically between them. This model also explains the anomalous behavior of the Scx allele of the flanking homeotic gene, Antennapedia. This allele, which is associated with an insertion near the Antennapedia P1 promoter, inactivates the Antennapedia P1 and P2 promoters in cis and derepresses the Scr promoters both in cis and on the homologous chromosome.     

Molecular organization of the cut locus of Drosophila melanogaster

Mutations of the cut locus (ct) of Drosophila can be divided into four groups based on their phenotypes and complementation patterns. Each group alters the phenotype of a different set of tissues. Two hundred kilobases of ct DNA, located in 7B1-2, have been cloned by chromosomal walking, and the cloned sequences have been used to analyze more than 40 mutants. Based on the location of transposable element mutations and the extent of deficiencies and an inversion, four cut locus regions can be defined. Mutations in each region affect the phenotype of a different set of tissues. The most centromere proximal region contains mutations that are null for cut locus function. Within individual regions, a higher level of organization can be detected.