methods-to-test-endocrine-disruption-in-drosophila-melanogaster

Methods to Test Endocrine Disruption in <em>Drosophila melanogaster</em>

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...

UniNa "Federico II"

We report here the genetic, molecular, and functional characterization of the Drosophila melanogaster minifly (mfl) gene. Genetic analysis shows that mfl is essential for Drosophila viability and fertility. While P-element induced total loss-of-function mutations cause lethality, mfl partial loss-of-function mutations cause pleiotropic defects, such as extreme reduction of body size, developmental delay, hatched abdominal cuticle, and reduced female fertility. Morphological abnormalities characteristic of apoptosis are found in the ovaries, and a proportion of eggs laid by mfl mutant females degenerates during embryogenesis. We show that mfl encodes an ubiquitous nucleolar protein that plays a central role in ribosomal RNA processing and pseudouridylation, whose known eukaryotic homologues are yeast Cfb5p, rat NAP57 and human dyskerin, encoded by the gene responsible for the X-linked dyskeratosis congenita disease. mfl genetic analysis represents the first in vivo functional characterization of a member of this highly conserved gene family from higher eukaryotes. In addition, we report that mfl hosts an intron encoded box H/ACA snoRNA gene, the first member of this class of snoRNAs identified so far from Drosophila.

minifly, a Drosophila gene required for ribosome biogenesis.

&#x5728;&#x771F;&#x6838;&#x7EC6;&#x80DE;&#xFF0C;&#x6BCF;&#x4E2A;&#x5206;&#x5B50;&#x7684; H/ACA &#x5C0F;&#x6838;&#x4EC1; RNA (&#x6838;&#x4EC1;&#x86CB;&#x767D;) &#x88C5;&#x914D;&#x4E0E;&#x56DB;&#x79CD;&#x8FDB;&#x5316;&#x4FDD;&#x6301;&#x7684;&#x6838;&#x5FC3;&#x86CB;&#x767D;&#x6765;&#x64B0;&#x5199;&#x5177;&#x4F53;&#x6838;&#x7CD6;&#x6838;&#x86CB;&#x767D;&#x9897;&#x7C92;&#x3002;&#x6709;&#x5047;&#x5C3F;&#x82F7;&#x5408;&#x9176;&#x6D3B;&#x6027;&#x548C;&#x50AC;&#x5316;&#x8F6C;&#x6362;&#x9009;&#x5B9A;&#x7684;&#x5C3F;&#x82F7;&#x4E3A;&#x5047;&#x5C3F;&#x82F7;&#x7684;&#x56DB;&#x4E2A;&#x6838;&#x5FC3;&#x7EC4;&#x4EF6;&#x4E4B;&#x4E00;&#x3002;&#x5047;&#x5C3F;&#x82F7;&#x5408;&#x6210;&#x9176;&#x5BB6;&#x65CF;&#x6210;&#x5458;&#x662F;&#x9AD8;&#x5EA6;&#x4FDD;&#x5B88;&#x7684;&#x3002;&#x9664;&#x50AC;&#x5316;&#x76EE;&#x6807; RNAs &#x7684; pseudouridylation&#xFF0C;&#x4ED6;&#x4EEC;&#x8FDB;&#x884C;&#x7684;&#x5404;&#x79CD;&#x57FA;&#x672C;&#x804C;&#x80FD;&#x6709;&#x5173;&#x6838;&#x7CD6;&#x4F53;&#x7684;&#x751F;&#x7269;&#x53D1;&#x751F;&#xFF0C;&#x5E76;&#x5728;&#x54FA;&#x4E73;&#x52A8;&#x7269;&#xFF0C;&#x7AEF;&#x7C92;&#x7EF4;&#x6301;&#x3002;&#x8FDB;&#x4E00;&#x6B65;&#x8C03;&#x67E5;&#x5047;&#x5C3F;&#x82F7;&#x5408;&#x6210;&#x9176;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x7684;&#x5206;&#x5B50;&#x673A;&#x5236;&#xFF0C;&#x6211;&#x4EEC;&#x5206;&#x6790;&#x4E86;&#x8FD9;&#x4E2A;&#x5BB6;&#x5EAD;&#x5F88;&#x8BE6;&#x7EC6;&#x7684;&#x679C;&#x8747;&#x6210;&#x5458;&#x7684;&#x8F6C;&#x5F55;&#x6D3B;&#x6027;&#x3002;

&#x7F16;&#x7801;/&#x975E;&#x7F16;&#x7801;&#x91CD;&#x53E0;&#x7684;&#x4F53;&#x7CFB;&#x7ED3;&#x6784;&#x7F16;&#x7801;&#x679C;&#x8747;&#x5047;&#x5C3F;&#x82F7;&#x5408;&#x9176;&#x7684;&#x57FA;&#x56E0;&#x3002;

In eukaryotic cells, each molecule of H/ACA small nucleolar RNA (snoRNA) assembles with four evolutionarily conserved core proteins to compose a specific ribonucleoprotein particle. One of the four core components has pseudouridine synthase activity and catalyzes the conversion of a selected uridine to pseudouridine. Members of the pseudouridine synthase family are highly conserved. In addition to catalyzing pseudouridylation of target RNAs, they carry out a variety of essential functions related to ribosome biogenesis and, in mammals, to telomere maintenance. To investigate further the molecular mechanisms underlying the expression of pseudouridine synthase genes, we analyzed the transcriptional activity of the Drosophila member of this family in great detail.

The coding/non-coding overlapping architecture of the gene encoding the Drosophila pseudouridine synthase.

Dans les cellules eucaryotes, chaque mol&eacute;cule de H/ACA petits ARN nucl&eacute;olaire (ARNpno) assemble avec quatre prot&eacute;ines de base &eacute;volutivement conserv&eacute;s pour composer une particule ribonucl&eacute;oprot&eacute;ique sp&eacute;cifiques. Une des quatre principales composantes a une activit&eacute; pseudouridine synthase et catalyse la conversion d&#39;un uridine s&eacute;lectionn&eacute; en pseudouridine. Membres de la famille des pseudouridine synthase sont tr&egrave;s conserv&eacute;s. En plus de catalyser la pseudouridylation des ARN cibles, qu&#39;ils exercent diverses fonctions essentielles li&eacute;es &agrave; la biog&eacute;n&egrave;se des ribosomes et, chez les mammif&egrave;res, &agrave; l&#39;entretien des t&eacute;lom&egrave;res. Pour &eacute;tudier davantage les m&eacute;canismes mol&eacute;culaires qui sous-tendent l&#39;expression des g&egrave;nes de pseudouridine synthase, nous avons analys&eacute; l&#39;activit&eacute; transcriptionnelle de la membre de drosophile de cette famille dans les moindres d&eacute;tails.

L&#39;architecture qui se chevauchent codage/non codante du g&egrave;ne codant pour la drosophile pseudouridine synthase.

In eukaryotischen Zellen baut jedes Molek&uuml;l H/ACA kleinen nucleolar RNS (SnoRNA) mit vier evolution&auml;r konservierte Kern-Proteine, eine spezifische Ribonucleoprotein Partikel zu komponieren. Einer der vier Kernkomponenten hat Pseudouridin-Synthase-Aktivit&auml;t und katalysiert die Umwandlung von einer ausgew&auml;hlten Uridin zu Pseudouridin. Pseudouridin Synthase Familienmitglieder sind stark konserviert. Zus&auml;tzlich zu katalysieren Pseudouridylierung des Ziels RNAs, f&uuml;hren sie eine Vielzahl von Aufgaben im Zusammenhang mit Ribosom Biogenese und bei S&auml;ugetieren Telomer-Wartung. Um die molekularen Mechanismen des Ausdrucks der Pseudouridin Synthase Gene weiter zu untersuchen, analysieren wir die transkriptionelle Aktivit&auml;t der Drosophila-Mitglied dieser Familie sehr detailliert.

Die Kodierung/nicht-kodierenden &uuml;berlappende Architektur gen f&uuml;r die Drosophila-Pseudouridin-Synthase.

Nelle cellule eucariotiche, ogni molecola di H/ACA small nucleolar RNA (snoRNA) Assembla con quattro proteine evolutionarily conserved core a comporre una particella specifica ribonucleoproteina. Uno dei componenti del quattro nucleo ha pseudouridine synthase attivit&agrave; e catalizza la conversione di un selezionato uridina in pseudouridine. Membri della famiglia pseudouridine synthase sono altamente conservati. Oltre a catalizzare pseudouridylation del target RNAs, che svolgono una variet&agrave; di funzioni essenziali relative a biogenesi del ribosoma e, nei mammiferi, al mantenimento dei telomeri. Per indagare ulteriormente i meccanismi molecolari sottostanti l&#39;espressione dei geni di pseudouridine synthase, abbiamo analizzato l&#39;attivit&agrave; trascrizionale del membro di questa famiglia in grande dettaglio della drosofila.

La codifica/non codificanti sovrapposta architettura del gene che codifica il drosofila pseudouridine synthase.

&#x771F;&#x6838;&#x751F;&#x7269;&#x306E;&#x7D30;&#x80DE;&#x3067;&#x306F;&#x3001;&#x5404;&#x5206;&#x5B50; H/ACA &#x5C0F;&#x3055;&#x306A;&#x6838;&#x5C0F;&#x4F53; RNA (snoRNA) &#x306E;&#x7279;&#x5B9A;&#x30EA;&#x30DC;&#x6838;&#x86CB;&#x767D;&#x8CEA;&#x7C92;&#x5B50;&#x3092;&#x69CB;&#x6210;&#x3059;&#x308B; 4 &#x3064;&#x306E;&#x9032;&#x5316;&#x7684;&#x4FDD;&#x5B58;&#x30B3;&#x30A2;&#x86CB;&#x767D;&#x8CEA;&#x3092;&#x7D44;&#x307F;&#x7ACB;&#x3066;&#x307E;&#x3059;&#x3002;4 &#x3064;&#x306E;&#x30B3;&#x30A2; &#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8;&#x306E;&#x3044;&#x305A;&#x308C;&#x304B;&#x306E; pseudouridine &#x5408;&#x6210;&#x9175;&#x7D20;&#x6D3B;&#x6027;&#x304C;&#x3042;&#x308B;&#x3057;&#x3001;&#x9078;&#x629E;&#x3057;&#x305F;&#x30A6;&#x30EA;&#x30B8;&#x30F3;&#x306E; pseudouridine &#x3078;&#x306E;&#x5909;&#x63DB;&#x3092;&#x89E6;&#x5A92;&#x3057;&#x307E;&#x3059;&#x3002;Pseudouridine &#x5408;&#x6210;&#x9175;&#x7D20;&#x306E;&#x5BB6;&#x65CF;&#x306E;&#x30E1;&#x30F3;&#x30D0;&#x30FC;&#x306F;&#x9AD8;&#x5EA6;&#x306B;&#x4FDD;&#x5B58;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;Pseudouridylation &#x6A19;&#x7684; Rna &#x306E;&#x793E;&#x4F1A;&#x3078;&#x306E;&#x8CA2;&#x732E;&#x306B;&#x52A0;&#x3048;&#x3066;&#x3001;&#x5F7C;&#x3089;&#x306F;&#x3055;&#x307E;&#x3056;&#x307E;&#x306A;&#x30EA;&#x30DC;&#x30BD;&#x30FC;&#x30E0;&#x306E;&#x751F;&#x5408;&#x6210;&#x3068;&#x3001;&#x54FA;&#x4E73;&#x985E;&#x3001;&#x30C6;&#x30ED;&#x30E1;&#x30A2;&#x7DAD;&#x6301;&#x306B;&#x5FC5;&#x8981;&#x4E0D;&#x53EF;&#x6B20;&#x306A;&#x6A5F;&#x80FD;&#x3092;&#x9042;&#x884C;&#x3057;&#x307E;&#x3059;&#x3002;Pseudouridine &#x5408;&#x6210;&#x9175;&#x7D20;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x767A;&#x73FE;&#x306E;&#x5206;&#x5B50;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x3055;&#x3089;&#x306B;&#x8ABF;&#x67FB;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x3053;&#x306E;&#x3059;&#x3070;&#x3089;&#x3057;&#x3044;&#x7D30;&#x90E8;&#x3067;&#x5BB6;&#x65CF;&#x306E;&#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8; &#x30E1;&#x30F3;&#x30D0;&#x30FC;&#x306E;&#x8EE2;&#x5199;&#x6D3B;&#x6027;&#x3092;&#x5206;&#x6790;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;

&#x7B26;&#x53F7;&#x5316;/&#x975E;&#x30B3;&#x30FC;&#x30C7;&#x30A3;&#x30F3;&#x30B0;&#x91CD;&#x8907;&#x30A2;&#x30FC;&#x30AD;&#x30C6;&#x30AF;&#x30C1;&#x30E3; &#x30B7;&#x30E7;&#x30A6;&#x30B8;&#x30E7;&#x30A6;&#x30D0;&#x30A8; pseudouridine &#x5408;&#x6210;&#x9175;&#x7D20;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x3002;

&#xC9C4; &#xD575; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; H/ACA &#xC791;&#xC740; nucleolar RNA (snoRNA)&#xC758; &#xAC01; &#xBD84;&#xC790;&#xC5D0;&#xB294; &#xD2B9;&#xC815; ribonucleoprotein &#xC785;&#xC790; &#xC791;&#xC131; 4 &#xC9C4;&#xD654; &#xC801;&#xC73C;&#xB85C; &#xBCF4;&#xC874;&#xB41C; &#xD575;&#xC2EC; &#xB2E8;&#xBC31;&#xC9C8; &#xC870;&#xB9BD;. 4 &#xAC00;&#xC9C0; &#xD575;&#xC2EC; &#xAD6C;&#xC131; &#xC694;&#xC18C; &#xC911; &#xD558;&#xB098; pseudouridine synthase &#xD65C;&#xB3D9;&#xC740; &#xD558; &#xACE0; &#xC120;&#xD0DD;&#xD55C; uridine pseudouridine &#xBCC0;&#xD658; catalyzes. Pseudouridine synthase &#xAC00;&#xC871;&#xC758; &#xACE0;&#xB3C4;&#xB85C; &#xBCF4;&#xC874; &#xB429;&#xB2C8;&#xB2E4;. &#xB300;&#xC0C1; Rnas&#xC758; pseudouridylation catalyzing &#xC774;&#xC678;&#xC5D0; &#xADF8;&#xB4E4;&#xC740; &#xB2E4;&#xC591; &#xD55C; &#xAD00;&#xB828; &#xB9AC;&#xBCF4;&#xC19C; &#xC18D; &#xBC0F; &#xD3EC;&#xC720;&#xB958;, telomere &#xC720;&#xC9C0; &#xAD00;&#xB9AC;&#xC5D0;&#xC5D0;&#xC11C; &#xD544;&#xC218;&#xC801;&#xC778; &#xAE30;&#xB2A5;&#xC744; &#xC218;&#xD589;. &#xCD94;&#xAC00; &#xC870;&#xC0AC; pseudouridine synthase &#xC720;&#xC804;&#xC790; &#xD45C;&#xD604;&#xC758; &#xAE30;&#xBCF8; &#xBD84;&#xC790; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;, &#xC6B0;&#xB9AC;&#xB294; &#xD6CC;&#xB96D;&#xD55C; &#xC138;&#xBD80; &#xC0AC;&#xD56D;&#xC5D0;&#xC11C;&#xC774; &#xAC00;&#xC871;&#xC758; &#xCD08;&#xD30C;&#xB9AC; &#xBA64;&#xBC84;&#xC758; transcriptional &#xD65C;&#xB3D9;&#xC744; &#xBD84;&#xC11D; &#xD588;&#xB2E4;.

&#xCF54;&#xB529;/&#xBE44; &#xCF54;&#xB529; &#xACB9;&#xCE58;&#xB294; &#xC544;&#xD0A4;&#xD14D;&#xCC98; Drosophila pseudouridine synthase &#xC778;&#xCF54;&#xB529; &#xC720;&#xC804;&#xC790;&#xC758;.

En las c&eacute;lulas eucariotas, cada mol&eacute;cula de ARN nucleolar peque&ntilde;o (snoRNA) de H/ACA monta con cuatro prote&iacute;nas evolutivamente conservado n&uacute;cleo para componer una part&iacute;cula de la Ribonucleoprote&iacute;na espec&iacute;ficos. Uno de los cuatro componentes tiene pseudouridina sintasa actividad y cataliza la conversi&oacute;n de un seleccionado Uridina en pseudouridina. Miembros de la familia de la sintasa de pseudouridina est&aacute;n muy conservados. Adem&aacute;s de catalizar pseudouridylation de destino RNAs, llevan a cabo una variedad de funciones esenciales relacionados con la biog&eacute;nesis de ribosomas y, en los mam&iacute;feros, al mantenimiento de los tel&oacute;meros. Para investigar m&aacute;s a fondo los mecanismos moleculares subyacentes a la expresi&oacute;n de genes de pseudouridina sintasa, analizamos la actividad transcripcional de la Drosophila este familiar con gran detalle.

La codificaci&oacute;n/no codificante superpuesta arquitectura del gene que codificaba la Drosophila pseudouridina sintasa.

BAP and PBAP constitute the two different forms of the Drosophila melanogaster Brahma chromatin remodelers. A common multisubunit core, containing the Brahma ATPase, can associate either with Osa to form the BAP complex or with Bap170, Bap180, and Sayp to constitute the PBAP complex. Although required for many biological processes, recent genetic analyses revealed that one role of the BAP complex during Drosophila wing development is the proper regulation of EGFR target genes. Here, we show that Bap170, a distinctive subunit of the PBAP complex, participates instead in the negative regulation of EGFR signaling. In adults, loss of Bap170 generates phenotypes similar to the defects induced by hyperactivation of the EGFR pathway, such as overrecruitment of cone and photoreceptor cells and formation extra veins. In genetic interactions, bap170 mutations suppress the loss of veins and photoreceptors caused by mutations affecting the activity of the EGFR pathway. Our results suggest a dual requirement of the PBAP complex: for transcriptional repression of rhomboid and for efficient expression of argos. Interestingly, genetic evidence also indicates that Bap170-mediated repression of rho is inhibited by EGFR signaling, suggesting a scenario of mutual antagonism between EGFR signaling and PBAP function.

Ennio Giordano

Department of Biology

UniNa "Federico II"

Methods to Test Endocrine Disruption in Drosophila melanogaster