Method for Labeling Transcripts in Individual <em>Escherichia coli</em> Cells for Single-molecule Fluorescence <em>In Situ</em> Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ ...

Weizmann Institute of Science

Previous studies suggest that cells within the CD34(+) hematopoietic stem cell compartment are endowed with immune regulatory activity. Furthermore, it is possible to expand the human regulatory cells upon short-term culture of purified CD34+ cells with an early-acting cytokine cocktail. We now show that addition of anti-CD28, anti-CD2, interleukin-2 (IL-2), anti-IL-10, or IL-12 to the bulk mixed lymphocyte reaction (MLR) cannot reverse the inhibitory activity of the CD34+ cells, ruling out anergy-based mechanisms or mechanisms involving Th1-Th2 skewing. Furthermore, phenotyping of cells present after addition of CD34+ cells to the bulk MLR ruled out potential induction of plasmacytoid dendritic precursors, known to be endowed with regulatory activity. In contrast, the inhibitory activity of CD34+ cells could be reversed by adding the caspase inhibitor BD-FMK to the bulk MLR, indicating a deletion-based mechanism. The deletion can be inhibited by anti-tumor necrosis factor-alpha (anti-TNF-alpha) and not by anti-transforming growth factor-beta (anti-TGF-beta), suggesting a potential role for TNF-alpha in the regulatory activity of CD34+ cells.

Immune regulatory activity of CD34+ progenitor cells: evidence for a deletion-based mechanism mediated by TNF-alpha.

Galectin-8, a mammalian beta-galactoside binding lectin, functions as an extracellular matrix protein that forms high affinity interactions with integrins. Here we demonstrated that soluble galectin-8 inhibits cell cycle progression and induces growth arrest. These effects cannot be attributed to interference with cell adhesion but can be attributed to a 4-5-fold increase in the cellular content of the cyclin-dependent kinase inhibitor p21, which was already evident following a 4-h incubation of H1299 cells with galectin-8. The increase in p21 levels was preceded by a 3-5-fold increase in JNK and protein kinase B (PKB) activities. Accordingly, SP600125, the inhibitor of JNK, and wortmannin, the inhibitor of phosphatidylinositol 3-kinase, which is the upstream activator of PKB, inhibited the increase in the cellular content of p21. Furthermore, overexpression of a dominant inhibitory form of SEK1, the upstream kinase regulator of JNK, inhibited both JNK activation and p21 accumulation. When p21 expression was inhibited by cycloheximide, galectin-8 directed the cells toward apoptosis, which involves induction of poly(ADP-ribose) polymerase cleavage. Indeed, galectin-8-induced apoptosis was 2-fold higher in HTC (p21-null) cells when compared with parental HTC cells. Because overexpression of galectin-8 attenuates the rate of DNA synthesis, stable colonies that overexpress and secrete galectin-8 can be generated only in cells overexpressing a growth factor receptor, such as the insulin receptor. These results implicate galectin-8 as a modulator of cellular growth through up-regulation of p21. This process involves activation of JNK, which enhances the synthesis of p21, combined with the activation of PKB, which inhibits p21 degradation. These effects of the lectin depended upon protein-sugar interactions and were induced when galectin-8 was present as a soluble ligand or when it was overexpressed in cells.

Cyclin-dependent kinase inhibitors and JNK act as molecular switches, regulating the choice between growth arrest and apoptosis induced by galectin-8.

The tumor suppressor BRCA2 protein plays a major role in the regulation of Rad51-catalyzed homologous recombination. BRCA2 interacts with monomeric Rad51 primarily via conserved BRC domains and coordinates the formation of Rad51 filaments at double-stranded DNA (dsDNA) breaks. A number of cancer-associated mutations in BRC4 and BRC2 domains have been reported. To elucidate their effects on homologous recombination, we studied Rad51 filament formation on single-stranded DNA and dsDNA substrates and Rad51-catalyzed strand exchange, in the presence of wild-type and mutated peptides of either BRC4 or BRC2. While the wild-type BRC2 and BRC4 peptides inhibited filament formation and, thus, strand exchange, the mutated forms decreased significantly these inhibitory effects. These results are consistent with a three-dimensional model for the interface between individual BRC repeats and Rad51. We suggest that mutations at sites crucial for the association between Rad51 and BRC domains impair the ability of BRCA2 to recruit Rad51 to dsDNA breaks, hampering recombinational repair.

Cancer-associated mutations in BRC domains of BRCA2 affect homologous recombination induced by Rad51.

Homologous recombination plays pivotal roles in DNA repair and in the generation of genetic diversity. To locate homologous target sequences at which strand exchange can occur within a timescale that a cell's biology demands, a single-stranded DNA-recombinase complex must search among a large number of sequences on a genome by forming synapses with chromosomal segments of DNA. A key element in the search is the time it takes for the two sequences of DNA to be compared, i.e. the synapse lifetime. Here, we visualize for the first time fluorescently tagged individual synapses formed by RecA, a prokaryotic recombinase, and measure their lifetime as a function of synapse length and differences in sequence between the participating DNAs. Surprisingly, lifetimes can be approximately 10 s long when the DNAs are fully heterologous, and much longer for partial homology, consistently with ensemble FRET measurements. Synapse lifetime increases rapidly as the length of a region of full homology at either the 3'- or 5'-ends of the invading single-stranded DNA increases above 30 bases. A few mismatches can reduce dramatically the lifetime of synapses formed with nearly homologous DNAs. These results suggest the need for facilitated homology search mechanisms to locate homology successfully within the timescales observed in vivo.

Caught in the act: the lifetime of synaptic intermediates during the search for homology on DNA.

&#x5728; DNA &#x4FEE;&#x590D;&#x548C;&#x4EE3;&#x9057;&#x4F20;&#x591A;&#x6837;&#x6027;&#x7684;&#x540C;&#x6E90;&#x91CD;&#x7EC4;&#x65B9;&#x9762;&#x53D1;&#x6325;&#x5173;&#x952E;&#x4F5C;&#x7528;&#x3002;&#x67E5;&#x627E;&#x540C;&#x6E90;&#x76EE;&#x6807;&#x5E8F;&#x5217;&#x4E0A;&#x7684;&#x94A2;&#x7EDE;&#x7EBF;&#x4EA4;&#x6362;&#x53EF;&#x4EE5;&#x53D1;&#x751F;&#x65F6;&#x95F4;&#x523B;&#x5EA6;&#x5185;&#x7EC6;&#x80DE;&#x751F;&#x7269;&#x5B66;&#x8981;&#x6C42;&#xFF0C;&#x5355;&#x94FE; DNA &#x91CD;&#x7EC4;&#x9176;&#x590D;&#x5408;&#x7269;&#x5FC5;&#x987B;&#x901A;&#x8FC7;&#x5F62;&#x6210;&#x7A81;&#x89E6;&#x4E0E;&#x67D3;&#x8272;&#x4F53; DNA &#x7247;&#x65AD;&#x641C;&#x7D22;&#x5927;&#x91CF;&#x7684;&#x57FA;&#x56E0;&#x7EC4;&#x5E8F;&#x5217;&#x4E4B;&#x95F4;&#x3002;&#x5728;&#x641C;&#x7D22;&#x4E2D;&#x7684;&#x4E00;&#x4E2A;&#x5173;&#x952E;&#x56E0;&#x7D20;&#x662F;&#x65F6;&#x95F4;&#xFF0C;&#x8BA9;&#x4E24;&#x4E2A;&#x5E8F;&#x5217;&#x7684; DNA &#x8FDB;&#x884C;&#x6BD4;&#x8F83;&#xFF0C;&#x5373;&#x7A81;&#x89E6;&#x751F;&#x5B58;&#x671F;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x53EF;&#x89C6;&#x5316;&#x9996;&#x6B21;&#x9057;&#x4F20;&#x7269;&#x8D28;&#x6807;&#x8BB0;&#x5355;&#x4E2A;&#x7A81;&#x89E6; RecA&#x3001; &#x539F;&#x6838;&#x91CD;&#x7EC4;&#x9176;&#xFF0C;&#x7531;&#x7EC4;&#x6210;&#x548C;&#x8861;&#x91CF;&#x5176;&#x751F;&#x5B58;&#x671F;&#x4F5C;&#x4E3A;&#x51FD;&#x6570;&#x7684;&#x7A81;&#x89E6;&#x957F;&#x5EA6;&#x548C;&#x5E8F;&#x5217;&#x4E2D;&#x53C2;&#x4E0E;&#x56FD;&#x5BB6;&#x4E3B;&#x7BA1;&#x90E8;&#x95E8;&#x4E4B;&#x95F4;&#x7684;&#x5DEE;&#x5F02;&#x3002;&#x51FA;&#x4EBA;&#x610F;&#x6599;&#x7684;&#x662F;&#xFF0C;&#x751F;&#x5B58;&#x671F;&#x53EF;&#x4EE5;&#x662F;&#x5927;&#x7EA6; 10 s &#x957F;&#x65F6;&#x56FD;&#x5BB6;&#x4E3B;&#x7BA1;&#x90E8;&#x95E8;&#x662F;&#x5B8C;&#x5168;&#x5F02;&#x6E90;&#xFF0C;&#x5E76;&#x4E3A;&#x90E8;&#x5206;&#x540C;&#x6E90;&#xFF0C;&#x4E00;&#x8D2F;&#x4E0E;&#x5408;&#x594F;&#x97F3;&#x54C1;&#x6D4B;&#x91CF;&#x957F;&#x5F97;&#x591A;&#x3002;&#x7A81;&#x89E6;&#x751F;&#x5B58;&#x671F;&#x4F5C;&#x4E3A;&#x4E00;&#x4E2A;&#x533A;&#x57DF;&#x7684;&#x5145;&#x5206;&#x540C;&#x6E90;&#x6027;&#x5728;&#x4EFB;&#x4E00;&#x957F;&#x5EA6;&#x8FC5;&#x901F;&#x589E;&#x52A0; 3&#39;-&#x6216; 5&#39; &#x7AEF;&#x7684;&#x5165;&#x4FB5;&#x7684;&#x5355;&#x94FE; DNA &#x9AD8;&#x4E8E; 30 &#x57FA;&#x5730;&#x3002;&#x51E0;&#x4E2A;&#x4E0D;&#x5339;&#x914D;&#x53EF;&#x4EE5;&#x5927;&#x5927;&#x51CF;&#x5C11;&#x7A81;&#x89E6;&#x5F62;&#x6210;&#x5177;&#x6709;&#x8FD1;&#x540C;&#x6E90; Dna &#x7684;&#x751F;&#x5B58;&#x671F;&#x3002;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x8868;&#x660E;&#xFF0C;&#x9700;&#x8981;&#x7684;&#x4FBF;&#x5229;&#x540C;&#x6E90;&#x6027;&#x641C;&#x7D22;&#x673A;&#x5236;&#x6765;&#x5B9A;&#x4F4D;&#x6210;&#x529F;&#x5730;&#x5728;&#x89C2;&#x5BDF;&#x5230;&#x4F53;&#x5185;&#x7684;&#x65F6;&#x95F4;&#x5C3A;&#x5EA6;&#x5185;&#x7684;&#x540C;&#x6E90;&#x6027;&#x3002;

&#x5F53;&#x573A;&#x6293;&#x83B7;&#xFF1A; &#x7A81;&#x89E6;&#x4E2D;&#x95F4;&#x4EA7;&#x7269;&#x5BF9; DNA &#x540C;&#x6E90;&#x6027;&#x641C;&#x7D22;&#x7684;&#x751F;&#x5B58;&#x671F;&#x3002;

Recombinaison homologue joue un r&ocirc;le essentiel dans la r&eacute;paration de l&#39;ADN et la g&eacute;n&eacute;ration de la diversit&eacute; g&eacute;n&eacute;tique. Pour localiser la cible homologue des s&eacute;quences &agrave; quelle Bourse brin peuvent se produire dans un d&eacute;lai que les demandes de biologie d&#39;une cellule, un complexe de recombinase-ADN monocat&eacute;naire doit rechercher parmi un grand nombre de s&eacute;quences sur un g&eacute;nome en formant des synapses avec des segments d&#39;ADN chromosomiques. Un &eacute;l&eacute;ment cl&eacute; dans la recherche est le temps que n&eacute;cessaire pour les deux s&eacute;quences d&#39;ADN &agrave; comparer, c&#39;est-&agrave;-dire la dur&eacute;e de vie de synapse. Nous visualisons ici, pour la premi&egrave;re fois fluorescent &eacute;tiquet&eacute;s synapses individuelles forment par RecA, une recombinase procaryote et mesurent leur dur&eacute;e de vie en fonction de la longueur de la synapse et diff&eacute;rences dans la s&eacute;quence entre les ADN des participants. &Eacute;tonnamment, les dur&eacute;es de vie peuvent &ecirc;tre environ 10 s long lorsque l&#39;ADN sont enti&egrave;rement h&eacute;t&eacute;rologue et beaucoup plus de temps pour l&#39;homologie partielle, en coh&eacute;rence avec les mesures de frette ensemble. Dur&eacute;e de vie de Synapse augmente rapidement la longueur d&#39;une r&eacute;gion d&#39;homologie compl&egrave;te soit la 3&#39;-ou 5&#39;-extr&eacute;mit&eacute;s de l&#39;ADN monocat&eacute;naire augmente au dessus de 30 bases envahissant. Quelques incompatibilit&eacute;s peuvent r&eacute;duire consid&eacute;rablement la dur&eacute;e de vie des synapses form&eacute;es avec presque homologues DNAs. Ces r&eacute;sultats sugg&egrave;rent la n&eacute;cessit&eacute; de m&eacute;canismes de recherche d&#39;homologie facilit&eacute;e localiser une homologie avec succ&egrave;s dans les d&eacute;lais observ&eacute;es in vivo.

Pris en flagrant d&eacute;lit : la dur&eacute;e de vie des interm&eacute;diaires synaptiques au cours de la recherche d&#39;homologie ADN.

Homologe Rekombination spielt entscheidende Rollen in der DNA-Reparatur und in der Generation der genetischen Vielfalt. Homologe Ziel finden k&ouml;nnen-Sequenzen an das Strang-Exchange in einem Zeitrahmen auftreten, dass eine Zelle Biologie verlangt, ein einzeln-angeschwemmte DNA-Recombinase Komplex unter eine gro&szlig;e Anzahl von Sequenzen auf ein Genom suchen muss, durch die Bildung von Synapsen mit chromosomaler DNA-Segmente. Ein Schl&uuml;sselelement bei der Suche ist die ben&ouml;tigte Zeit f&uuml;r die beiden Sequenzen von DNA verglichen werden, d.h. die Synapse-Lebensdauer. Hier, visualisieren wir zum ersten Mal Leuchtstoff markierte einzelne Synapsen durch RecA, einer prokaryotischen Recombinase gebildet und ihre Lebensdauer als Funktion der Synapse L&auml;nge und Unterschiede in der Sequenz zwischen den teilnehmenden DNAs zu messen. &Uuml;berraschend Lebenszeiten k&ouml;nnen sein etwa 10 s lang, wenn die DNAs voll heterologe und viel l&auml;nger f&uuml;r partiellen Homologie, konsequent mit Ensemble FRET Messungen sind. Synapse Lebensdauer erh&ouml;ht sich schnell als die L&auml;nge einer Region voller Homologie entweder der 3&#39;-oder 5&#39;-Enden von den eindringenden einzelstr&auml;ngige DNA &uuml;ber 30 Basen erh&ouml;ht. Ein paar Unterschiede k&ouml;nnen die Lebensdauer der Synapsen gebildet mit fast homologe DNAs reduzieren. Diese Resultate schlagen die Notwendigkeit einer erleichterten Homologie Suchmechanismen, Homologie erfolgreich in dem Zeitrahmen beobachtet in-vivo zu finden.

Caught in the Act: die Lebensdauer der synaptischen Zwischenprodukte bei der Suche nach Homologie auf DNA.

Ricombinazione omologa interpreta ruoli cardine nella riparazione del DNA e nella generazione della diversit&agrave; genetica. Per individuare la destinazione omologhe sequenze in cui exchange strand possono verificarsi entro un lasso di tempo che esige biologia di una cella, un single-stranded DNA-recombinase complesso deve cercare tra un gran numero di sequenze in un genoma formando sinapsi con segmenti cromosomiche del DNA. Un elemento chiave nella ricerca &egrave; il tempo che necessario per le due sequenze di DNA da confrontare, cio&egrave; la durata di sinapsi. Qui, noi visualizzare per la prima volta fluorescente contrassegnati singole sinapsi formano da RecA, un procariote ricombinasi e misurano la loro vita in funzione della lunghezza di sinapsi e differenze nella sequenza tra il DNAs partecipanti. Sorprendentemente, la vite possono essere circa 10 s lunga quando il DNAs sono completamente eterologa e molto pi&ugrave; tempo per omologia parziale, coerentemente con le misurazioni di FRET ensemble. Durata della sinapsi aumenta rapidamente come la lunghezza di una regione di omologia pieno sia il 3&#39;-o 5&#39;-estremit&agrave; di invasori single-stranded DNA aumenta sopra 30 basi. Alcune incongruenze possono ridurre drasticamente la durata della sinapsi formate con DNAs quasi omologhe. Questi risultati suggeriscono la necessit&agrave; di meccanismi di ricerca di omologia agevolato individuare omologia con successo entro i tempi osservati in vivo.

Caught in the act: la durata di synaptic intermedi durante la ricerca di omologia sul DNA.

&#x76F8;&#x540C;&#x7D44;&#x63DB;&#x3048; DNA &#x4FEE;&#x5FA9;&#x3068;&#x907A;&#x4F1D;&#x7684;&#x591A;&#x69D8;&#x6027;&#x306E;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x76F8;&#x540C;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x3092;&#x691C;&#x7D22;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x306F;&#x3001;&#x9396;&#x306E;&#x4EA4;&#x63DB;&#x3067;&#x3001;&#x30BF;&#x30A4;&#x30E0; &#x30B9;&#x30B1;&#x30FC;&#x30EB;&#x5185;&#x7D30;&#x80DE;&#x306E;&#x751F;&#x7269;&#x5B66;&#x8981;&#x6C42;&#x3057;&#x3066;&#x3044;&#x308B;&#x3001;DNA &#x306E;&#x67D3;&#x8272;&#x4F53;&#x306E;&#x30BB;&#x30B0;&#x30E1;&#x30F3;&#x30C8;&#x3092;&#x6301;&#x3064;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x3092;&#x5F62;&#x6210;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x308A;&#x3001;&#x4E00;&#x672C;&#x9396; DNA &#x30B7;&#x30E5;&#x30D7;&#x30EA;&#x30F3;&#x30AC;&#x30FC;&#x8907;&#x96D1;&#x306A;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x3092;&#x30B2;&#x30CE;&#x30E0;&#x4E0A;&#x306E;&#x591A;&#x6570;&#x306E;&#x9593;&#x3067;&#x691C;&#x7D22;&#x3059;&#x308B;&#x5FC5;&#x8981;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x767A;&#x751F;&#x3057;&#x307E;&#x3059;&#x3002;&#x691C;&#x7D22;&#x306E;&#x91CD;&#x8981;&#x306A;&#x8981;&#x7D20;&#x304C; DNA &#x306E; 2 &#x3064;&#x306E;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x306E;&#x305F;&#x3081;&#x3001;&#x3059;&#x306A;&#x308F;&#x3061;&#x3001;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x306E;&#x6709;&#x52B9;&#x671F;&#x9593;&#x3092;&#x6BD4;&#x8F03;&#x3059;&#x308B;&#x6642;&#x9593;&#x3067;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x521D;&#x3081;&#x3066;&#x86CD;&#x5149;&#x30BF;&#x30B0;&#x500B;&#x3005; &#x306E;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x306F;&#x539F;&#x6838;&#x751F;&#x7269;&#x306E;&#x30EA;&#x30B3;&#x30F3;&#x30D3;&#x30CA;&#x30FC;&#x30BC; RecA &#x306B;&#x3088;&#x3063;&#x3066;&#x5F62;&#x6210;&#x3055;&#x308C;&#x305F;&#x3001;&#x4E00;&#x751F;&#x306E;&#x9593;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x306E;&#x9577;&#x3055;&#x3068;&#x53C2;&#x52A0;&#x306E; Dna &#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x76F8;&#x9055;&#x306E;&#x95A2;&#x6570;&#x3068;&#x3057;&#x3066;&#x6E2C;&#x5B9A;&#x3092;&#x53EF;&#x8996;&#x5316;&#x3057;&#x307E;&#x3059;&#x3002;&#x9A5A;&#x3044;&#x305F;&#x3053;&#x3068;&#x306B;&#x3001;&#x6709;&#x52B9;&#x671F;&#x9593;&#x304C;&#x7D04; 10 &#x3092;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x5B8C;&#x5168;&#x306B;&#x7570;&#x7A2E;&#x3068;&#x306F;&#x308B;&#x304B;&#x306B;&#x9577;&#x3044;&#x30D5;&#x30EC;&#x30C3;&#x30C8;&#x89B3;&#x6E2C;&#x3068;&#x4E00;&#x8CAB;&#x3057;&#x3066;&#x3001;&#x90E8;&#x5206;&#x7684;&#x306A;&#x76F8;&#x540C;&#x6027;&#x306E; Dna &#x3059;&#x308B;&#x5834;&#x5408;&#x9577;&#x3044; s&#x3002;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x5BFF;&#x547D;&#x304C;&#x98DB;&#x8E8D;&#x7684;&#x306B;&#x5897;&#x5927;&#x306B;&#x304A;&#x3051;&#x308B;&#x76F8;&#x540C;&#x3067;&#x3044;&#x305A;&#x308C;&#x304B;&#x306E;&#x9818;&#x57DF;&#x306E;&#x9577;&#x3055; 3&#39;-&#x307E;&#x305F;&#x306F; 5&#39;-&#x4FB5;&#x5165;&#x306E;&#x4E00;&#x672C;&#x9396; DNA &#x304C;&#x5897;&#x52A0; 30 &#x62E0;&#x70B9;&#x4EE5;&#x4E0A;&#x306E;&#x7AEF;&#x3092;&#x3002;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x4E0D;&#x4E00;&#x81F4;&#x306F;&#x3001;&#x5927;&#x5E45;&#x306B;&#x307B;&#x307C;&#x76F8;&#x540C; Dna &#x3067;&#x5F62;&#x6210;&#x3055;&#x308C;&#x308B;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x306E;&#x6709;&#x52B9;&#x671F;&#x9593;&#x3092;&#x6E1B;&#x3089;&#x3059;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;&#x76F8;&#x540C; in vivo &#x89B3;&#x5BDF;&#x30B9;&#x30B1;&#x30FC;&#x30EB;&#x304C;&#x6B63;&#x5E38;&#x306B;&#x5185;&#x3092;&#x691C;&#x7D22;&#x3059;&#x308B;&#x5BB9;&#x6613;&#x76F8;&#x540C;&#x6027;&#x691C;&#x7D22;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x306E;&#x305F;&#x3081;&#x306E;&#x5FC5;&#x8981;&#x6027;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;

&#x884C;&#x70BA;&#x306B;&#x5DFB;&#x304D;&#x8FBC;&#x307E;&#x308C;&#x308B;: dna &#x76F8;&#x540C;&#x6027;&#x691C;&#x7D22;&#x4E2D;&#x306B;&#x30B7;&#x30CA;&#x30D7;&#x30B9;&#x306E;&#x4E2D;&#x9593;&#x4F53;&#x306E;&#x6709;&#x52B9;&#x671F;&#x9593;&#x3002;

&#xB3D9;&#xC885; &#xC7AC;&#xC870;&#xD569; DNA &#xC218;&#xB9AC;&#xC640; &#xC720;&#xC804;&#xC801; &#xB2E4;&#xC591;&#xC131;&#xC758; &#xC138;&#xB300; &#xC911;&#xCD94;&#xC801;&#xC778; &#xC5ED;&#xD560;&#xC744; &#xD55C;&#xB2E4;. &#xB3D9;&#xC885; &#xB300;&#xC0C1; &#xCC3E;&#xC744; &#xC2DC;&#xD000;&#xC2A4;&#xB294; &#xC2A4;&#xD2B8;&#xB79C;&#xB4DC; exchange&#xC5D0;&#xC11C; &#xC138;&#xD3EC;&#xC758; &#xC0DD;&#xBB3C;&#xD559; &#xC694;&#xAD6C; &#xB2E8;&#xC77C; &#xAC00;&#xB2E5; DNA recombinase &#xBCF5;&#xC7A1; &#xD55C; DNA&#xC758; &#xC5FC;&#xC0C9;&#xCCB4; &#xC138;&#xADF8;&#xBA3C;&#xD2B8;&#xC640; &#xC2DC; &#xB0C5; &#xC2A4;&#xB97C; &#xD615;&#xC131; &#xD558; &#xC5EC; &#xB9CE;&#xC740; &#xAC8C;&#xB188; &#xC2DC;&#xD000;&#xC2A4; &#xC911;&#xC5D0;&#xC11C; &#xAC80;&#xC0C9; &#xD574;&#xC57C; &#xD569;&#xB2C8;&#xB2E4; &#xD45C;&#xC2DC;&#xC904; &#xB0B4;&#xC5D0;&#xC11C; &#xBC1C;&#xC0DD;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xAC80;&#xC0C9;&#xC758; &#xD575;&#xC2EC; &#xC694;&#xC18C;, &#xC989; &#xB0C5; &#xC218;&#xBA85; &#xBE44;&#xAD50; DNA&#xC758; &#xB450; &#xC2DC;&#xD000;&#xC2A4;&#xC5D0; &#xB300; &#xD55C; &#xAC78;&#xB9AC;&#xB294; &#xC2DC;&#xAC04;&#xC785;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC;&#xAC00; &#xCC98;&#xC74C;&#xC73C;&#xB85C; &#xBD99;&#xC77C; &#xD0DC;&#xADF8;&#xAC00; &#xAC1C;&#xBCC4; &#xB0C5; RecA, prokaryotic recombinase&#xC5D0; &#xC758;&#xD574; &#xD615;&#xC131; &#xD558; &#xACE0; &#xC790;&#xC2E0;&#xC758; &#xC77C;&#xC0DD; &#xB0C5; &#xAE30;&#xAC04;&#xACFC; &#xCC38;&#xC5EC; DNAs &#xC21C;&#xC11C; &#xCC28;&#xC774;&#xC758; &#xD568;&#xC218;&#xB85C; &#xCE21;&#xC815;&#xC5D0; &#xB300; &#xD55C; &#xC2DC;&#xAC01;&#xD654;. &#xB180;&#xB78D;&#xAC8C;&#xB3C4;, &#xC218;&#xBA85; &#xC57D; 10 &#xC218; s&#xB294; Dnas&#xB294; &#xC644;&#xBCBD; &#xD558; &#xAC8C; &#xBD84;&#xB9AC; &#xD558; &#xACE0; &#xC559;&#xC0C1;&#xBE14; &#xBB34;&#xC11C; &#xC6CC; &#xCE21;&#xC815;&#xC744; &#xC77C;&#xAD00; &#xB418; &#xAC8C; &#xBD80;&#xBD84; homology&#xB97C; &#xC704;&#xD55C; &#xD6E8;&#xC52C; &#xB354; &#xAE34;. &#xB0C5; &#xC218;&#xBA85; &#xC601;&#xC5ED;&#xC5D0;&#xC11C; &#xC804;&#xCCB4; homology &#xAE38;&#xC774;&#xB85C; &#xAE09;&#xC18D; &#xD558; &#xAC8C; &#xC99D;&#xAC00; 3&#39;-&#xB610;&#xB294; 5&#39;-&#xB05D;&#xC758; &#xB2E8;&#xC77C; &#xAC00;&#xB2E5; DNA &#xC99D;&#xAC00; 30 &#xAE30;&#xC9C0; &#xC704;&#xC5D0; &#xCE68;&#xC785;. &#xBA87; &#xAC00;&#xC9C0; &#xBD88;&#xC77C;&#xCE58; &#xB0C5; &#xAC70;&#xC758; &#xB3D9;&#xC885; Dnas&#xC640; &#xD615;&#xC131;&#xC758; &#xC218;&#xBA85;&#xC744; &#xD68D;&#xAE30;&#xC801;&#xC73C;&#xB85C; &#xC904;&#xC77C; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xACB0;&#xACFC; timescales &#xAD00;&#xCC30; &#xC0DD;&#xCCB4; &#xC870;&#xAC74; &#xB0B4;&#xC5D0;&#xC11C; &#xC131;&#xACF5;&#xC801;&#xC73C;&#xB85C; homology&#xB97C; &#xCC3E;&#xC73C;&#xB824;&#xACE0; &#xCD09;&#xC9C4;&#xB41C; homology &#xAC80;&#xC0C9; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC694;&#xC131;&#xC744; &#xC81C;&#xC548; &#xD569;&#xB2C8;&#xB2E4;.

&#xD589;&#xC704;&#xC5D0;&#xC11C; &#xBD99;&#xC7A1;&#xB294;: homology DNA&#xC5D0; &#xB300; &#xD55C; &#xAC80;&#xC0C9; &#xD558;&#xB294; &#xB3D9;&#xC548; &#xC2DC; &#xB0C5; &#xC2A4; &#xC911;&#xAC04;&#xCCB4;&#xC758; &#xC218;&#xBA85;.

Recombinaci&oacute;n hom&oacute;loga juega un papel fundamental en la reparaci&oacute;n del ADN y en la generaci&oacute;n de diversidad gen&eacute;tica. Para localizar el destino hom&oacute;logo secuencias en que cambio de filamento pueden ocurrir en un plazo que exige la biolog&iacute;a de la c&eacute;lula, un complejo de ADN-Recombinasa monocatenario debe buscar entre un gran n&uacute;mero de secuencias en un genoma mediante la formaci&oacute;n de sinapsis con segmentos cromos&oacute;micos del ADN. Un elemento clave en la b&uacute;squeda es el tiempo que tarda para las dos secuencias de ADN para comparar, es decir, la vida &uacute;til de la sinapsis. Aqu&iacute;, visualizamos por primera vez fluorescencia etiquetas sinapsis individuales forman por RecA, una Recombinasa procariota y medir su vida en funci&oacute;n de la longitud de la sinapsis y las diferencias en la secuencia entre los DNAs participantes. Sorprendentemente, la vida puede ser aproximadamente 10 s largo cuando los DNAs son completamente heter&oacute;loga y mucho m&aacute;s para la homolog&iacute;a parcial, consistentemente con las mediciones de traste de conjunto. Vida de sinapsis aumenta r&aacute;pidamente como la longitud de una regi&oacute;n de homolog&iacute;a completo en cualquiera los 3&#39;-5&#39; finales o de los invasores ADN monocatenario aumenta por encima de 30 bases. Algunos desajustes pueden reducir dram&aacute;ticamente la vida de sinapsis formada con casi hom&oacute;logos DNAs. Estos resultados sugieren la necesidad de mecanismos de b&uacute;squeda de homolog&iacute;a facilitado localizar homolog&iacute;a con &eacute;xito dentro de los plazos observados in vivo.

Sorprendido en el acto: la vida &uacute;til de productos intermedios sin&aacute;pticas durante la b&uacute;squeda de homolog&iacute;a de ADN.

Cell-to-cell variations in protein abundance, called noise, give rise to phenotypic variability between isogenic cells. Studies of noise have focused on stochasticity introduced at transcription, yet the effects of post-transcriptional regulatory processes on noise remain unknown. We study the effects of RyhB, a small-RNA of Escherichia coli produced on iron stress, on the phenotypic variability of two of its downregulated target proteins, using dual chromosomal fusions to fluorescent reporters and measurements in live individual cells. The total noise of each of the target proteins is remarkably constant over a wide range of RyhB production rates despite cells being in stress. In fact, coordinate downregulation of the two target proteins by RyhB reduces the correlation between their levels. Hence, an increase in phenotypic variability under stress is achieved by decoupling the expression of different target proteins in the same cell, rather than by an increase in the total noise of each. Extrinsic noise provides the dominant contribution to the total protein noise over the total range of RyhB production rates. Stochastic simulations reproduce qualitatively key features of our observations and show that a feed-forward loop formed by transcriptional extrinsic noise, an sRNA and its target genes exhibits strong noise filtration capabilities.

Effects of post-transcriptional regulation on phenotypic noise in Escherichia coli.

The inherently stochastic nature of biomolecular processes is one of the main sources giving rise to cell-to-cell variations in protein and mRNA abundance, termed noise. Noise in isogenic populations can enhance survival under adverse conditions and stress, and has therefore played a fundamental role in evolution. On the other hand, noise may have detrimental effects and therefore cells must also display robustness to fluctuations and possess mechanisms of control in order to function properly. Noise can be introduced at every step in the cascade of intermediate events resulting in the production of functional proteins. While initial studies of noise focused on stochasticity introduced at the transcriptional level, recent years have witnessed a gradual shift of emphasis into the effects that post-transcriptional processes have on phenotypic noise. Here, we survey the insights that have been gained on the effects of processes that modify RNA transcript populations on phenotypic noise, including regulation by noncoding RNAs in prokaryotes and eukaryotes, alternative splicing and transcriptional interference.

Phenotypic noise: effects of post-transcriptional regulatory processes affecting mRNA.

The search for specific sequences on long genomes is a key process in many biological contexts. How can specific target sequences be located with high efficiency, within physiologically relevant times? We addressed this question for viral integration, a fundamental mechanism of horizontal gene transfer driving prokaryotic evolution, using the infection of Escherichia coli bacteria with bacteriophage λ and following the establishment of a lysogenic state. Following the targeting process in individual live E. coli cells in real time revealed that λ DNA remains confined near the entry point of a cell following infection. The encounter between the 15-bp-long target sequence on the chromosome and the recombination site on the viral genome is facilitated by the directed motion of bacterial DNA generated during chromosome replication, in conjunction with constrained diffusion of phage DNA. Moving the native bacterial integration site to different locations on the genome and measuring the integration frequency in these strains reveals that the frequencies of the native site and a site symmetric to it relative to the origin are similar, whereas both are significantly higher than when the integration site is moved near the terminus, consistent with the replication-driven mechanism we propose. This novel search mechanism is yet another example of the exquisite coevolution of λ with its host.

Location of the unique integration site on an Escherichia coli chromosome by bacteriophage lambda DNA in vivo.

Under nitrogen deprivation, filaments of the cyanobacterium Anabaena undergo a process of development, resulting in a one-dimensional pattern of nitrogen-fixing heterocysts separated by about ten photosynthetic vegetative cells. Many aspects of gene expression before nitrogen deprivation and during the developmental process remain to be elucidated. Furthermore, the coupling of gene expression fluctuations between cells along a multicellular filament is unknown. We studied the statistics of fluctuations of gene expression of HetR, a transcription factor essential for heterocyst differentiation, both under steady-state growth in nitrogen-rich conditions and at different times following nitrogen deprivation, using a chromosomally-encoded translational hetR-gfp fusion. Statistical analysis of fluorescence at the individual cell level in wild-type and mutant filaments demonstrates that expression fluctuations of hetR in nearby cells are coupled, with a characteristic spatial range of circa two to three cells, setting the scale for cellular interactions along a filament. Correlations between cells predominantly arise from intercellular molecular transfer and less from cell division. Fluctuations after nitrogen step-down can build up on those under nitrogen-replete conditions. We found that under nitrogen-rich conditions, basal, steady-state expression of the HetR inhibitor PatS, cell-cell communication influenced by the septal protein SepJ and positive HetR auto-regulation are essential determinants of fluctuations in hetR expression and its distribution along filaments. A comparison between the expression of hetR-gfp under nitrogen-rich and nitrogen-poor conditions highlights the differences between the two HetR inhibitors PatS and HetN, as well as the differences in specificity between the septal proteins SepJ and FraC/FraD. Activation, inhibition and cell-cell communication lie at the heart of developmental processes. Our results show that proteins involved in these basic ingredients combine together in the presence of inevitable stochasticity in gene expression, to control the coupled fluctuations of gene expression that give rise to a one-dimensional developmental pattern in this organism.

Spatial fluctuations in expression of the heterocyst differentiation regulatory gene hetR in Anabaena filaments.

Post-transcriptional regulatory processes may change transcript levels and affect cell-to-cell variability or noise. We study small-RNA downregulation to elucidate its effects on noise in the iron homeostasis network of Escherichia coli In this network, the small-RNA RyhB undergoes stoichiometric degradation with the transcripts of target genes in response to iron stress. Using single-molecule fluorescence in situ hybridization, we measured transcript numbers of the RyhB-regulated genes sodB and fumA in individual cells as a function of iron deprivation. We observed a monotonic increase of noise with iron stress but no evidence of theoretically predicted, enhanced stoichiometric fluctuations in transcript numbers, nor of bistable behavior in transcript distributions. Direct detection of RyhB in individual cells shows that its noise is much smaller than that of these two targets, when RyhB production is significant. A generalized two-state model of bursty transcription that neglects RyhB fluctuations describes quantitatively the dependence of noise and transcript distributions on iron deprivation, enabling extraction of in vivo RyhB-mediated transcript degradation rates. The transcripts' threshold-linear behavior indicates that the effective in vivo interaction strength between RyhB and its two target transcripts is comparable. Strikingly, the bacterial cell response exhibits Fur-dependent, switch-like activation instead of a graded response to iron deprivation.

Rinat Arbel-Goren

Department of Physics of Complex Systems

Weizmann Institute of Science

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

Rinat Arbel-Goren

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Physics of Complex Systems

Weizmann Institute of Science

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

Department of Physics of Complex Systems