Lethal(1)fibrillardysgenesis (I(1)fdg), a mutation affecting muscle development in the embryo of Drosophila melanogaster

The I(1)fdg mutation demonstrates two separate phases of lethality, depending on developmental conditions. At 32–33°C, an embryonic lethality is expressed whereas at lower temperatures a larval-pupal lethality is observed. This larval-pupal lethality characteristically produces noncondensed, curved puparia, and since the contraction of the pupa depends on strong muscular contraction, this phase of lethality implicates some involvement of abnormal musculature. The embryonic expression of I(1)fdg at 32–33°C is the subject of this study. In these embryos, which are alive but immobile (incapable of hatching), the fibrillar organization and fiber morphology of the somatic musculature varies from being apparently normal to being grossly abnormal. While the abnormalities appear as unusual distributions of fiber organelles, abnormal convolutions of the muscle fibers, and disorganizations of fibrillar components, it seems most probable that the underlying defect ultimately responsible resides in some system essential for Z body alignment and sarcomere formation. Accompanying the embryonic lethality, certain abnormalities in midgut development are observed which at present do not appear to be related to the defects observed in the somatic muscle.     

Fertility of Drosophila melanogaster females affected by mutation l(2)M167 DTS

We studied the fertility of D. melanogaster females heterozygous for the dominant temperature sensitive mutation l(2)M167 ^DTS , which exerts a recessive lethal effect at 25°C, under the conditions of stable temperature regimes 25, 28, and 29°C and changing regimes 25 → 29°C and 29 → 25°C. It was shown that inhibition of total activity of oogenesis due to a decreased number of functioning ovarioles is one of the mechanisms underlying the decreased fertility of l(2)M167 ^DTS /+ females. Analysis of individual fertility of each female confirmed also the role of sterility as a component of fertility of the females. Sterilization was realized due both to full depletion of functioning ovarioles and disturbed mechanism of laying the mature eggs onto a substrate as a result of violation of the feedback blocking normal ovulation, which led to the breakdown of ovarioles and filling of the abdominal cavity with mature oocytes. A significant polymorphism of heterozygous females by their fertility was observed. The intensity of sterilization and mortality of l(2)M167 ^DTS /+ females sharply increased at an elevated temperature (29°C), especially at the pupal stage.     

On the mechanism underlying the divergent retinal and bristle defects of M8* (E(spl)D) in Drosophila

Our results, using endogenous mutants and Gal4‐UAS driven transgenes, implicate multisite phosphorylation in repression by E(spl)M8. We propose that these phosphorylations occur in the morphogenetic furrow (MF) to reverse an auto‐inhibited state of M8, enabling repression of Atonal during R8 specification. Our studies address the paradoxical behavior of M8*, the truncated protein encoded by E(spl)D. We suggest that differences in N signaling in the bristle versus the eye underlie the antimorphic activity of M8* in N⁺ (ectopic bristles) and hypermorphic activity in N^spl (reduced eye). Ectopic M8* impairs eye development (in N^spl) only during establishment of the atonal feedback loop (anterior to the MF), but is ineffective after this time point. In contrast, a CK2 phosphomimetic M8 lacking Groucho (Gro) binding, M8SDΔGro, acts antimorphic in N⁺ and suppresses the eye/R8 and bristle defects of N^spl, as does reduced dosage of E(spl) or CK2. Multisite phosphorylation could serve as a checkpoint to enable a precise onset of repression, and this is bypassed in M8*. Additional implications are discussed. genesis 47:456–468, 2009. © 2009 Wiley‐Liss, Inc.     

Rm62, a DEAD‐box RNA helicase,complexes with DSP1 in Drosophila embryos

Two main classes of proteins, Polycomb group (PcG) and Trithorax group (TrxG), play a key role in the regulation of homeotic genes. These proteins act in multimeric complexes to remodel chromatin. A third class of proteins named Enhancers of Trithorax and Polycomb (ETP) modulates the activity of TrxG and PcG, but their role remains largely unknown. We previously identified an HMGB‐like protein, DSP1 (Dorsal Switch Protein 1), which was classified as an ETP. Preliminary studies have revealed that DSP1 is involved in multimeric complexes. Here we identify a DEAD‐box RNA helicase, Rm62, as partner of DSP1 in a 250‐kDa complex. Coimmunoprecipitation assays performed on embryo extracts indicate that DSP1 and Rm62 are associated in 3‐ to 12‐h embryos. Furthermore, DSP1 and Rm62 colocalize on polytene chromosomes. Consistent with these results, a mutation in Rm62 enhances a null mutation of dsp1 and also mutations of trxG or PcG, suggesting that Rm62 has characteristics of an ETP. We show here for the first time that an RNA helicase is involved in the maintenance of homeotic genes. genesis 48:244–253, 2010. © 2010 Wiley‐Liss, Inc.     

The persistence of short‐term cold acclimation in Drosophila melanogaster (Diptera: Drosophilidae)

Daily and seasonal fluctuations in temperature present significant challenges for the survival of many ectothermic species that can be tempered via thermal acclimation. In the present study, we use multiple naturally derived genotypes of Drosophila melanogaster to determine the persistence of beneficial short‐term thermal acclimation on subsequent survival after cold shock. We found that the benefit of short‐term acclimation persisted for 2 h in most genotypes after a rapid cold hardening treatment. Genotype did not directly influence the persistence of short‐term acclimation benefits, indicating that environmental variation may be more important for the persistence of acclimation benefits rather than genetic capacity for acclimation. The present study extends the current understanding of the limits and importance of short‐term acclimation events, providing greater detail on the timing of the loss of short‐term acclimation benefits in a genetically variable natural population.     

Tumor suppression in Drosophila is causally related to the function of the lethal(2)tumorous imaginal discs gene,a dnaJ homolog

The Drosophila melanogaster tumor suppressor gene lethal(2)tumorous imaginal discs (l(2)tid) causes in homozygotes malignant growth of cells of the imaginal discs and the death of the mutant larvae at the time of puparium formation. We describe the molecular cloning of the 1(2)tid+ gene and its temporal expression pattern in the wild-type and mutant alleles. Germ line rescue of the tumor phenotype was achieved with a 7.0 kb Hindlll-fragment derived from the polytene chromosome band 59F5. The l(2)tid+ gene spans approximately 2.5 kb of genomic DNA. The protein coding region, 1,696 bps long, is divided by an intron into two exons. The predicted Tid⁵⁶ protein contains 518 amino acids and possesses a theoretical molecular weight of 56 kDa. It shows significant homology to all known DnaJ related proteins from bacteria, yeast, and man. The possible function of the Tid⁵⁶ protein in tumor suppression is delineated. © 1995 Wiley-Liss, Inc.     

Pathological role of a point mutation (T315I) in BCR‐ABL1 protein—A computational insight

BCR‐ABL protein is one of the most potent target to treat chronic myeloid leukemia (CML). Apart from other mutations, T315I is especially challenging as it confers resistance to all first‐ and second‐generation tyrosine kinase inhibitors. So, a thorough study of altered behavior upon mutation is crucially needed. To understand the resistance mechanism of mutant BCR‐ABL protein, we organized a long‐term molecular dynamics simulation (500 ns) and performed the detailed comparative conformational analysis. We found that due to mutation at 315th position (threonine to isoleucine), original structures deviated from normal, and attained a flexible conformation. Our observations pave a clear path toward designing new inhibitors against resistant BCR‐ABL1 protein and suggest a strategy where additional flexibility governed by mutation could be given an appropriate consideration.     

四环素与3,4-苯并芘复合污染对抗性基因tetA(C)产生高抗性突变的影响

【目的】探究环境中同时存在低浓度四环素(tetracycline,TC)和3,4-苯并芘(benzo [a] pyrene,Bap)对抗性基因tetA(C)产生高抗性突变的影响。【方法】以大肠杆菌(Escherichia coli,E. coli)为宿主菌株,pACYC184质粒作为载体,四环素抗性基因tetA(C)作为研究对象,采用易错PCR构建基因文库的方法,建立基因突变位点对应高抗性的关系密码表。同时设置添加低浓度TC且添加0–30 mg/L Bap以及仅添加0–30 mg/L Bap的处理组,培养携带pACYC184质粒的大肠杆菌14 d,每组中随机挑选10株获得高抗性的菌株,对其中的tetA(C)基因片段进行测序,再结合突变位点密码表,计算高抗性菌株中由基因突变产生高抗性菌株的比例。【结果】测序结果显示在低浓度TC选择压力下,Bap浓度越高时,高抗性基因突变株占的比例也越高(P≤0.01),而不添加TC时,Bap浓度与高抗性基因突变株占比之间无变化规律(P0.05)。【结论】当环境中同时存在Bap和低浓度TC时,高抗性突变基因易于通过选择压力保存下来。     

Variation of within‐crop microhabitat use by Drosophila suzukii (Diptera: Drosophilidae) in blackberry

Drosophila suzukii Matsumara (Diptera: Drosophilidae) is an invasive vinegar fly that infests ripe and ripening soft skinned fruits. In the south‐eastern United States, blackberry (Rubus spp.) crops are heavily impacted by D. suzukii, and current management tactics rely on the use of broad‐spectrum insecticides targeted to adult populations. An improved understanding of D. suzukii biology and ecology are necessary to create sustainable management options. Knowledge of how D. suzukii interacts with resources will enable targeted management actions in the future. In this present study, we monitored larval infestation throughout the blackberry canopy and found that infestation was highest in the inner portion of the canopy and lower in more exposed locations. We also documented higher humidity within the cane canopy relative to the edge of the field. A difference in abiotic conditions may create within‐crop microhabitats that D. suzukii is able to exploit. Future research will explore how to take advantage of these microhabitats in pest management programs.     

The Drosophila ribosomal protein L14-encoding gene, identified by a novel Minute mutation in a dense cluster of previously undescribed genes in cytogenetic region 66D

The Minute phenotype results from mutations at >50 loci scattered throughout the genome of Drosophila. Common traits of the Minute phenotype are short and thin bristles, slow development, and recessive lethality. Here, we report a novel P-element induced Minute mutation, P{lacW}M(3)66D ¹ , that maps to region 66D on chromosome 3L. Flies heterozygous for P{lacW}M(3)66D ¹ have a strong Minute phenotype. Molecular characterisation of the chromosomal region revealed three previously undescribed Drosophila genes clustered within a 5-kb genomic fragment. Two of the genes have significant sequence homology to genes for the mammalian ribosomal proteins L14 and RD, respectively, and share a joint 240-bp promoter region harbouring the P-element insert. Quantitative Northern blot analyses showed the mutation to affect RPL14 mRNA levels only. Interestingly, the reduction in abundance of RPL14 mRNA is not constitutive, indicating that the promoter function abolished by the inserted P-element is utilised with different efficiencies in different developmental situations. Remobilisation of the P element produced wild-type flies with normal levels of RPL14 mRNA, demonstrating that the mutant phenotype is caused by the insertion. P{lacW}M(3)66D ¹ joins a growing list of Minute mutations associated with ribosomal protein-haploinsufficiency. Received: 20 January 1997 / Accepted: March 3 1997     

JIL-1 kinase, a member of the male-specific lethal (MSL) complex, is necessary for proper dosage compensation of eye pigmentation in Drosophila

The upregulation of the JIL-1 kinase on the male X chromosome and its association with the male-specific lethal (MSL) complex suggest that JIL-1 may play a role in regulating dosage compensation. To directly test this hypothesis we measured eye pigment levels of mutants in the X-linked white gene in an allelic series of JIL-1 hypomorphic mutants. We show that dosage compensation of w(a) alleles that normally do exhibit dosage compensation was severely impaired in the JIL-1 mutant backgrounds. As a control we also examined a hypomorphic white allele w(e) that fails to dosage compensate in males due to a pogo element insertion. In this case the relative pigment level measured in males as compared to females remained approximately the same even in the most severe JIL-1 hypomorphic background. These results indicate that proper dosage compensation of eye pigment levels in males controlled by X-linked white alleles requires normal JIL-1 function.     

Modulatory effects of Tabebuia impetiginosa (Lamiales, Bignoniaceae) on doxorubicin-induced somatic mutation and recombination in Drosophila melanogaster

The wing Somatic Mutation and Recombination Test (SMART) in D. melanogaster was used to study genotoxicity of the medicinal plant Tabebuia impetiginosa. Lapachol (naphthoquinone) and β-lapachone (quinone) are the two main chemical constituents of T. impetiginosa. These compounds have several biological properties. They induce apoptosis by generating oxygen-reactive species, thereby inhibiting topoisomerases (I and II) or inducing other enzymes dependent on NAD(P)H:quinone oxidoreductase 1, thus affecting cell cycle checkpoints. The SMART was used in the standard (ST) version, which has normal levels of cytochrome P450 (CYP) enzymes, to check the direct action of this compound, and in the high bioactivation (HB) version, which has a high constitutive level of CYP enzymes, to check for indirect action in three different T. impetiginosa concentrations (10%, 20% or 40% w/w). It was observed that T. impetiginosa alone did not modify the spontaneous frequencies of mutant spots in either cross. The negative results observed prompted us to study this phytotherapeuticum in association with the reference mutagen doxorubicin (DXR). In co-treated series, T. impetiginosa was toxic in both crosses at higher concentration, whereas in the HB cross, it induced a considerable potentiating effect (from ~24.0 to ~95.0%) on DXR genotoxity. Therefore, further research is needed to determine the possible risks associated with the exposure of living organisms to this complex mixture.     

Spaghetti,a homolog of human RPAP3 (RNA polymerase II‐associated protein 3), determines the fate of germline stem cells in Drosophila ovary

The Drosophila ovary provides an attractive model for studying the extrinsic or intrinsic factors that regulate the fate of germline stem cells (GSCs). Using this model, we identified a new role for Drosophila spaghetti (spag), encoding a homolog of human RNA polymerase II‐associated protein 3 (RPAP3), in regulating the fate of ovarian GSCs. Results from spag knockdown and genetic mosaic studies suggest that spag functions as an intrinsic factor for GSCs maintenance. Loss of Spag by, either spag RNAi or null mutation failed to trigger apoptosis in ovarian GSCs. Overexpression of spag led to negligible increases in the number of GSC/Cystoblast (CB) cells, suggesting that an excess of Spag is not sufficient to accelerate the proliferation of GSCs or delay CBs’ differentiation. Our study provides evidence supporting that spag is involved in adult stem cells maintenance. In addition, the RNAi screen results showed that knockdown of Hsp90, one of known Spag interacting partners, led to loss of ovarian GSCs in Drosophila. Heterozygous mutations in hsp90 (hsp90/+) dramatically accelerated the GSC loss in spag RNAi ovaries, suggesting that the Spag‐contained complex possibly plays an essential role in controlling the GSCs fate.     

Identification of a new mutation in Escherichia coli that suppresses a pbpB (Ts) phenotype in the presence of penicillin-binding protein 1B

Analysis of the functional role of penicillin-binding protein 1B (PBP1B) of Escherichia coli led us to find a new mutation able to suppress thermosensitive growth of the pbpB2158(Ts) mutant strain, which harbors a thermosensitive PBP3 protein only in the presence of a ponB+ background. The mutation, originally isolated in a strain with a high dosage of PBP1B, could also suppress the pbpB(Ts) phenotype when a single copy of the ponB gene was introduced. These results clearly give further support to the implication of PPB1B in the septation process in Escherichia coli.     

CRAC channel is inhibited by neomycin in a Ptdlns(4,5)P2‐independent manner

Depletion of intracellular Ca²⁺ stores evokes store‐operated Ca²⁺ entry through the Ca²⁺ release‐activated Ca²⁺ (CRAC) channels. In this study, we found that the store‐operated Ca²⁺ entry was inhibited by neomycin, an aminoglycoside that strongly binds phosphatidylinositol 4,5‐bisphosphate (PtdIns(4,5)P2). Patch clamp recordings revealed that neomycin blocked the CRAC currents reconstituted by co‐expression of Orai1 and Stim1 in HEK293 cells. Using a rapamycin‐inducible PtdIns(4,5)P2‐specific phosphatase (Inp54p) system to manipulate the PtdIns(4,5)P2 in the plasma membrane, we found that the CRAC current was not altered by PtdIns(4,5)P2 depletion. This result suggests that PtdIns(4,5)P2 is not required for CRAC channel activity, and thereby, neomycin inhibits CRAC channels in a manner that is independent of neomycin–PtdIns(4,5)P2 binding. Copyright © 2015 John Wiley & Sons, Ltd.