発表者：鈴庄

Genetics 161:721-731 (June 2002)

Ectopic Expression of the Drosophila Cdk1 Inhibitory Kinases, Wee1 and Myt1, Interferes With the Second Mitotic Wave and Disrupts Pattern Formation During Eye Development Donald M. Price, Zhigang Jin, Simon Rabinovitch and Shelagh D. Campbell1

(Abstract)
Wee1 kinases catalyze inhibitory phosphorylation of the mitotic regulator Cdk1, preventing mitosis during S phase and delaying it in response to DNA damage or developmental signals during G2. Unlike yeast, metazoans have two distinct Wee1-like kinases, a nuclear protein (Wee1) and a cytoplasmic protein (Myt1). We have isolated the genes encoding Drosophila Wee1 and Myt1 and are using genetic approaches to dissect their functions during normal development. Overexpression of Dwee1 or Dmyt1 during eye development generates a rough adult eye phenotype. The phenotype can be modified by altering the gene dosage of known regulators of the G2/M transition, suggesting that we could use these transgenic strains in modifier screens to identify potential regulators of Wee1 and Myt1. To confirm this idea, we tested a collection of deletions for loci that can modify the eye overexpression phenotypes and identified several loci as dominant modifiers. Mutations affecting the Delta/Notch signaling pathway strongly enhance a GMR-Dmyt1 eye phenotype but do not affect a GMR-Dwee1 eye phenotype, suggesting that Myt1 is potentially a downstream target for Notch activity during eye development. We also observed interactions with p53, which suggest that Wee1 and Myt1 activity can block apoptosis.

発表者：中島

Functional analysis of mutant and wild-type Drosophila origin recognition complex

PNAS October 9.2001 vol.98 no.21 11997-12002

(Abstract)
The origin recognition complex (ORC) is the DNA replication initiator protein in eukaryotes. We have reconstituted a functional recombinant Drosophila ORC and compared activities of the wild-type and several mutant ORC variants. Drosophila ORC is an ATPase, and our studies show that the ORC1 subunit is essential for ATP hydrolysis and for ATP-dependent DNA binding. Moreover, DNA binding by ORC reduces its ATP hydrolysis activity. In vitro, ORC binds to chromatin in an ATP-dependent manner, and this process depends on the functional AAA+ nucleotide-binding domain of ORC1. Mutations in the ATP-binding domain of ORC1 are unable to support cell-free DNA replication. However, mutations in the putative ATP-binding domain of either the ORC4 or ORC5 subunits do not affect either of these functions. We also provide evidence that the Drosophila ORC6 subunit is directly required for all of these activities and that a large pool of ORC6 is present in the cytoplasm, cytologically proximal to the cell membrane. Studies reported here provide the first functional dissection of a metazoan initiator and highlight the basic conserved and divergent features among Drosophila and budding yeast ORC complexes.

発表者：Shun-Chern Tsaur
Reed, L., and T. A. Markow 2004. Early events in speciation: Polymorphism
for hybrid male sterility in Drosophila PNAS 101:9009-9012.

Abstract
Capturing the process of speciation early enough to determine the initial genetic causes of reproductive isolation remains a major challenge in evolutionary biology. We have found, to our knowledge, the first example of substantial intraspecific polymorphism for genetic factors contributing to hybrid male sterility. Specifically, we show that the occurrence of hybrid male sterility in crosses between Drosophila mojavensis and its sister species, Drosophila arizonae, is controlled by factors present at different frequencies in different populations of D. mojavensis. In addition, we show that hybrid male sterility is a complex phenotype; some hybrid males with motile sperm still cannot sire offspring. Because male sterility factors in hybrids between these species are not yet fixed within D. mojavensis, this system provides an invaluable opportunity to characterize the genetics of reproductive isolation at an early stage.

発表者：土屋


Drosophila mitochondrial transcription factor b2 regulates mitochondrial DNA copy number and transcription in schneider cells. Matsushima Y, Garesse R, Kaguni LS.

(Abstract)
We report the cloning and molecular analysis of Drosophila mitochondrial transcription factor B2 (d-mt-TFB2), a protein that plays a role in mitochondrial transcription and mitochondrial DNA (mtDNA) replication in Drosophila. An RNA interference (RNAi) construct was designed that reduces expression of d-mtTFB2 to 5% of its normal level in Schneider cells. RNAi knock-down of d-mtTFB2 reduces the abundance of specific mitochondrial RNA transcripts 2- to 8-fold and decreases the copy number of mtDNA approximately 3-fold. In a corollary manner, we find that overexpression of d-mtTFB2 increases both the abundance of mitochondrial RNA transcripts and the copy number of mtDNA. In a comparative experiment, we find that overexpression of Drosophila mitochondrial transcription factor A (d-TFAM) increases mtDNA copy number with no significant effect on mitochondrial transcripts. This argues for a direct role for mtTFB2 in mitochondrial transcription and suggests that, if TFAM serves a role in transcription, its endogenous level limits mtDNA copy number but not transcription. Furthermore, we suggest that mtTFB2 increases mtDNA copy number by increasing the frequency of initiation of DNA replication, whereas TFAM serves to stabilize and package mtDNA in mitochondrial nucleoids. Our work represents the first study to document the function of mtTFB2 in vivo, establishing a dual role in regulation of both transcription and replication, and provides a benchmark for comparative biochemical studies in various animal systems.

発表者：木村

A conditional tissue-specific transgene expression system using inducible GAL4. Thomas Osterwalder, Kenneth S. Yoon, Benjamin H. White, and Haig Keshishian
PNAS October 23, 2001 vol. 98 12596-12601

(Abstract)
In Drosophila, the most widely used system for generating spatially restricted transgene expression is based on the yeast GAL4 protein and its target upstream activating sequence (UAS). To permit temporal as well as spatial control over UAS-transgene expression, we have explored the use of a conditional RU486-dependent GAL4 protein (GeneSwitch) in Drosophila. By using cloned promoter fragments of the embryonic lethal abnormal vision gene or the myosin heavy chain gene, we have expressed GeneSwitch specifically in neurons or muscles and show that its transcriptional activity within the target tissues depends on the presence of the activator RU486 (mifepristone). We used available UAS-reporter lines to demonstrate RU486-dependent tissuespecific transgene expression in larvae. Reporter protein expression could be detected 5 h after systemic application of RU486 by either feeding or "larval bathing." Transgene expression levels were dose-dependent on RU486 concentration in larval food, with low background expression in the absence of RU486. By using genetically altered ion channels as reporters, we were able to change the physiological properties of larval bodywall muscles in an RU486-dependent fashion. We demonstrate here the applicability of GeneSwitch for conditional tissue-specific expression in Drosophila, and we provide tools to control pre- and postsynaptic expression of transgenes at the larval neuromuscular junction during postembryonic life.

発表者：山口
Development. 2004 May;131(9):2089-99.

Sequence requirements for function of the Drosophila chorion gene locus ACE3 replicator and ori-beta origin elements. Zhang H, Tower J.

(Abstract)
The developmentally regulated amplification of the Drosophila third chromosome chorion gene locus requires multiple chromosomal elements. Amplification control element third chromosome (ACE3) appears to function as a replicator, in that it is required in cis for the activity of nearby DNA replication origin(s). Ori-beta is the major origin in the locus, and is a sequence-specific element that is sufficient for high-level amplification in combination with ACE3. Sequence requirements for amplification were examined using a transgenic construct that was buffered from chromosomal position effects by flanking insulator elements. The parent construct supported 18- to 20-fold amplification, and contained the 320 bp ACE3, the approximately 1.2 kb S18 chorion gene and the 840 bp ori-beta. Deletion mapping of ACE3 revealed that an evolutionarily conserved 142 bp core sequence functions in amplification in this context. Several deletions had quantitative effects, suggesting that multiple, partially redundant elements comprise ACE3. S. cerevisiae ARS1 origin sequences could not substitute for ori-beta, thereby confirming the sequence specificity of ori-beta. Deletion mapping of ori-beta identified two required components: a 140 bp 5' element and a 226 bp A/T-rich 3' element called the beta-region that has significant homology to ACE3. Antibody to the origin recognition complex subunit 2 (ORC2) recognizes large foci that localize to the endogenous chorion gene loci and to active transgenic constructs at the beginning of amplification. Mutations in Orc2 itself, or the amplification trans regulator satin eliminated the ORC2 foci. By contrast, with a null mutation of chiffon (dbf4-like) that eliminates amplification, diffuse ORC2 staining was still present, but failed to localize into foci. The data suggest a novel function for the Dbf4-like chiffon protein in ORC localization. Chromosomal position effects that eliminated amplification of transgenic constructs also eliminated foci formation. However, use of the buffered vector allowed amplification of transgenic constructs to occur in the absence of detectable foci formation. Taken together, the data suggest a model in which ACE3 and ori-beta nucleate the formation of a ORC2-containing chromatin structure that spreads along the chromosome in a mechanism dependent upon chiffon.

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発表者：松林
Distinct Roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA Silencing Pathways.

Young Sik Lee, Kenji Nakahara, John W. Pham, Kevin Kim, Zhengying He, Erik J. Sontheimer, and Richard W. Carthew

Cell, Vol. 117, 69-81, 2004

−siRNA/miRNAによる遺伝子サイレンシングにおける、Dicer-1とDicer-2での異なった役割−

The RNase III enzyme Dicer processes RNA into si-RNAs and miRNAs, which direct a RNA-induced si-lencing complex (RISC) to cleave mRNA or block its translation (RNAi). We have characterized mutationsin the Drosophila dicer-1 and dicer-2 genes. Mutationin dicer-1 blocks processing of miRNA precursors,whereas dicer-2 mutants are defective for processing siRNA precursors. It has been recently found that Dro-sophila Dicer-1 and Dicer-2 are also components of siRNA-dependent RISC (siRISC). We find that Dicer-1 and Dicer-2 are required for siRNA-directed mRNAcleavage, though the RNase III activity of Dicer-2 is not required. Dicer-1 and Dicer-2 facilitate distinct steps in the assembly of siRISC. However, Dicer-1 but notDicer-2 is essential for miRISC-directed translation repression. Thus, siRISCs and miRISCs are differentwith respect to Dicers in Drosophila.

発表者：加藤
Genes Dev. 2004 Jun 1;18(11):1251-62. Epub 2004 May 14.
A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin.

Schotta G, Lachner M, Sarma K, Ebert A, Sengupta R, Reuter G, Reinberg D, Jenuwein T.

Research Institute of Molecular Pathology (IMP), The Vienna Biocenter, A-1030 Vienna, Austria.

発表者：福井
Curr Biol. 2001 Jul 10;11(13):1017-27

Double-stranded RNA-mediated silencing of genomic tandem repeats and
transposable elements in the D. melanogaster germline.

Aravin AA, Naumova NM, Tulin AV, Vagin VV, Rozovsky YM, Gvozdev VA.

Department of Animal Molecular Genetics, Institute of Molecular
Genetics, 123182, Moscow, Russia.

BACKGROUND:
The injection of double-stranded RNA (dsRNA) has been shown to induce a potent sequence-specific inhibition of gene function in diverse invertebrate and vertebrate species. The homology-dependent posttranscriptional gene silencing (PTGS) caused by the introduction of transgenes in plants may be mediated by dsRNA. The analysis of Caenorhabditis elegans mutants impaired with dsRNA-mediated silencing and studies in plants implicate a biological role of dsRNA-mediated silencing as a transposon-repression and antiviral mechanism.
RESULTS:
We investigated the silencing of testis-expressed Stellate genes by paralogous Su(Ste) tandem repeats, which are known to be involved in the maintenance of male fertility in Drosophila melanogaster. We found that both strands of repressor Su(Ste) repeats are transcribed, producing sense and antisense RNA. The Stellate silencing is associated with the presence of short Su(Ste) RNAs. Cotransfection experiments revealed that Su(Ste) dsRNA can target and eliminate Stellate transcripts in Drosophila cell culture. The short fragment of Stellate gene that is homologous to Su(Ste) was shown to be sufficient to confer Su(Ste)-dependent silencing of a reporter construct in testes. We demonstrated that Su(Ste) dsRNA-mediated silencing affects not only Stellate expression but also the level of sense Su(Ste) RNA providing a negative autogenous regulation of Su(Ste) expression. Mutation in the spindle-E gene relieving Stellate silencing also leads to a derepression of the other genomic tandem repeats and retrotransposons in the germline.
CONCLUSIONS:
Homology-dependent gene silencing was shown to be used to inhibit Stellate gene expression in the D.melanogaster germline, ensuring male fertility. dsRNA-mediated silencing may provide a basis for negative autogenous control of gene expression. The related surveillance system is implicated to control expression of retrotransposons in the germline.

発表者：大迫
A Flagellar Polycystin-2 Homolog Required for Male Fertility in Drosophila Terry J. Watnick, Ying Jin, Erika Matunis, Maurice J. Kernan, and Craig Montell
Current Biology 13, 2179-2184 (2003)

-多発性嚢胞腎の原因遺伝子PKD2のショウジョウバエホモログは精子の雌貯精器官への移動に必要である-

発表者：都丸
Seasonal behavior in Drosophila melanogaster requires the
photoreceptors, the circadian clock, and phospholipase C.
B. H. Collins, E. Rosato and C. P. Kyriacou PNAS 101: 1945-1950 (2004)

温度や光環境によって影響を受けるlocomotor activityと、cry（cryptochrome: 青色感受性光受容体）、per（period: 時計）、norpA（no-receptor-potential-A: フォスフォリパーゼＣ）との関わりについて

ABSTRACT
Drosophila melanogaster locomotor activity responds to different seasonal conditions by thermosensitive regulation of splicing of a 3' intron in the period mRNA transcript. Here we demonstrate that the control of locomotor patterns by this mechanism is primarily light-dependent at low temperatures. At warmer temperatures, when it is vitally important for the fly to avoid midday desiccation, more stringent regulation of splicing is observed, requiring the light input received through the visual system during the day and the circadian clock at night. During the course of this study, we observed that a mutation in the no-receptor-potential-AP41 (norpAP41) gene, which encodes phospholipase-C, generated an extremely high level of 3' splicing. This cannot be explained simply by the mutation's effect on the visual pathway and suggests that norpAP41 is directly involved in thermosensitivity

発表者；橋本
SPECIFIC ASSEMBLY PATHWAY OF SARCOGLYCANS IS DEPENDENT
ON BETA- AND DELTA-SARCOGLYCAN.

WEIXING SHI, MSc,1 ZAILI CHEN, MD,1 JODI SCHOTTENFELD, BA,1
RICHARD C. STAHL, BSc,1 LOUIS M. KUNKEL, PhD,2 and YIU-MO CHAN,
サルコグリカン複合体の集合化について

Muscle Nerve 29: 409-419, 2004

(Abstract)
Mutations in sarcoglycans (SG) have been reported to cause autosomal-recessive limb-girdle muscular dystrophy (LGMD) and dilated cardiomyopathy. In skeletal and cardiac muscle, sarcoglycans exist as a complex of four transmembrane proteins (
-, -, -, and -SG). In this study,the assembly of the sarcoglycan complex was examined in a heterologous expression system. Our results demonstrated that the assembly process occurs as a discrete stepwise process. We found that -SG appears to play an initiating role and its association with -SG is essential for the proper localization of the sarcoglycan complex to the cell membrane. The incorporationof
-SG into the sarcoglycan complex occurs at the final stage by interaction with -SG. These findings were supported by chemical crosslinking of endogenous sarcoglycans in cultured myotubes. We have also provided evidence that glycosylation-defective mutations in -SG and a common mutation in -SG (C283Y) disrupt sarcoglycan-complex formation.
Our proposed model for the assembly and structure of sarcoglycans should generate important insight into their function in muscle as well as their role in muscular dystrophies and cardiomyopathies.

発表者：尾崎
J Neurosci. 2003 Nov 19;23(33):10495-502.
Dopamine and octopamine differentiate between aversive and appetitive olfactory memories in Drosophila.

Schwaerzel M, Monastirioti M, Scholz H, Friggi-Grelin F, Birman S, Heisenberg M.

Lehrstuhl fur Genetik und Neurobiologie, Biozentrum, Am Hubland, D-97074
Wurzburg, Germany.

The catecholamines play a major role in the regulation of behavior. Here we investigate, in the fly Drosophila melanogaster, the role of dopamine and octopamine (the presumed arthropod homolog of norepinephrine) during the formation of appetitive and aversive olfactory memories. We find that for the formation of both types of memories, cAMP signaling is necessary and sufficient within the same subpopulation of mushroom-body intrinsic neurons. On the other hand, memory formation can be distinguished by the requirement for different catecholamines, dopamine for aversive and octopamine for appetitive conditioning. Our results suggest that in associative conditioning, different memories are formed of the same odor under different circumstances, and that they are linked to the respective motivational systems by their specific modulatory pathways

発表者：従二
Excitatory and Inhibitory Switches for Courtship in the Brain of Drosophila melanogaster
Susan J. Broughton, Toshihiro Kitamoto, and Ralph J. Greenspan
Current Biology, Vol. 14, 538−547, 2004

−キイロショウジョウバエ脳の興奮性および抑制性求愛行動スイッチ−

SUMMARY
Background: Courtship is the best-studied behavior in Drosophila melanogaster, and work on its anatomical basis has concentrated mainly on the functional identification of sexually dimorphic sites in the brain. Much less is known of the more expansive, nondimorphic, but nonetheless essential, neural elements subserving male courtship behavior.
Results: Sites in the CNS mediating initiation and early steps of male courtship in Drosophila melanogaster were identified by analyzing the behavior of mosaic flies expressing transgenes designed either to suppress neurotransmission or enhance neuronal excitability. Suppression of neurotransmission was accomplished by means of the dominantly acting, temperature-sensitive dynamin mutation shibirets1, whereas enhanced neuronal excitability was produced by means of a novel, dominantly acting, truncated eag potassium channel. By using a new, landmark-based procedure for aligning diverse expression patterns among the various mosaic strains, a comparison of courtship performance and affected brain sites in strains expressing the transgenes identified a cluster of cells in the posterior lateral protocerebrum that exerts reciprocal effects on the initiation of courtship, suppressing it when they are inactivated and enhancing it when they are hyperactivated, indicative of cells that normally play an excitatory, triggering role. A separate group of nearby cells, slightly more anterior in the lateral protocerebrum, was found to inhibit courtship when its activity is enhanced, indicative of an inhibitory role in courtship.
Conclusions: A cluster of cells, some excitatory and some inhibitory, in the lateral protocerebrum regulates courtship initiation in Drosophila.
These cells are likely to be an integration center for the multiple sensory inputs that trigger male courtship.