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Validation of shRNA clones for gene silencing in 293FT cells

von Wen Wang

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[1.] Ww/Fragment 011 01 - Diskussion
Zuletzt bearbeitet: 2014-10-29 07:42:22 Hindemith
Fragment, Gesichtet, Hammond 2005, SMWFragment, Schutzlevel sysop, Verschleierung, Ww

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SleepyHollow02
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Untersuchte Arbeit:
Seite: 11, Zeilen: 1-10
Quelle: Hammond 2005
Seite(n): 5824, Zeilen: r.col: 20ff
[Drosha is a Class II enzyme. This enzyme assumes a pseudo-dimer catalytic] core similar to Dicer (Han et al, 2004). The substrates of Drosha, miRNA primary transcripts, are structurally distinct from Dicer substrates. Drosha does not process from a dsRNA terminus. Rather, data suggests that primarily the stem–loop structure is recognized. In particular, the loop size appears to be important for recognition (Zeng et al, 2005). In addition, unstructured sequences flanking the stem–loop are essential for processing (Chen et al, 2004; Zeng et al, 2005). It is not evident how Drosha is able to recognize these sequences, as they are outside of the dsRNA stem. Possibly other unidentified cofactors play a role. Conserved sequence elements have been found in flanking regions of C. elegans miRNAs (Ohler et al, 2004). Drosha is a Class II enzyme as shown in Fig. 1. This enzyme assumes a pseudo-dimer catalytic core similar to Dicer [27]. The substrate of Drosha, microRNA primary transcripts, is structurally distinct from Dicer substrates. Drosha does not process from a dsRNA terminus. Rather, data suggests that the stem–loop structure is recognized. In particular, the loop size appears to be important for recognition [28]. In addition, unstructured sequences flanking the stem–loop are essential for processing [29,30]. It is not evident how Drosha would recognize these sequences, as they are outside of the dsRNA stem. Possibly other, unidentified cofactors play a role. Conserved sequence elements have been found in flanking regions of C. elegans microRNAs [31].

[27] Han, J., Lee, Y., Yeom, K.H., Kim, Y.K., Jin, H. and Kim, V.N. (2004) The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev. 18, 3016–3027.

[28] Zeng, Y., Yi, R. and Cullen, B.R. (2005) Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha. EMBO J. 24, 138–148.

[29] Chen, C.Z., Li, L., Lodish, H.F. and Bartel, D.P. (2004) MicroRNAs modulate hematopoietic lineage differentiation. Science 303, 83–86.

[30] Zeng, Y. and Cullen, B.R. (2005) Efficient processing of primary microRNA hairpins by Drosha requires flanking non-structured RNA sequences. J. Biol. Chem..

[31] Ohler, U., Yekta, S., Lim, L.P., Bartel, D.P. and Burge, C.B. (2004) Patterns of flanking sequence conservation and a characteristic upstream motif for microRNA gene identification. RNA 10, 1309–1322

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Sichter
(SleepyHollow02), Hindemith


[2.] Ww/Fragment 011 11 - Diskussion
Zuletzt bearbeitet: 2014-10-29 17:06:55 Hindemith
Fragment, Geley and Mueller 2004, Gesichtet, SMWFragment, Schutzlevel sysop, Verschleierung, Ww

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Quelle: Geley and Mueller 2004
Seite(n): 987, Zeilen: l.col: 1ff
1.2.1.2 Execution phase: assembly of siRNA containing silencing complexes

Dicer-generated siRNAs are then incorporated into a large multiprotein complex, which is involved in various gene-silencing modes, and is called the RNA induced silencing complex, or RISC (Hammond et al, 2000; Nykanen et al, 2001). Processing of dsRNA and assembly of a functional RISC likely occurs in the cytoplasm, as Dicer is a cytosolic enzyme and RISC activity can be purified from the cytosol (Billy et al, 2001). R2D2, a Drosophila gene related to the C. elegans RNAi gene RDE-4, has been implicated in the transfer of siRNAs into the RISC (Liu et al, 2003). Generation of siRNAs from dsRNA in Drosophila embryo extracts, unwinding of the siRNA duplex, and incorporation into the RISC requires ATP (Nykanen et al, 2001). In contrast, human Dicer does not seem to rely on ATP for processing of dsRNA into siRNA molecules (Zhang et al., 2002).

2.2. The execution phase: assembly of siRNA containing silencing complexes

Dicer-generated siRNAs are then incorporated into a large multiprotein complex, which is involved in various gene-silencing modes, and is called the RNA induced silencing complex, or RISC (Hammond et al., 2000; Nykanen et al., 2001). Processing of dsRNA and assembly of a functional RISC likely occurs in the cytoplasm, as Dicer is a cytosolic enzyme and RISC activity can be purified from cytosol (Billy et al., 2001). R2D2, a Drosophila gene related to the C. elegans RNAi gene RDE-4, has been implicated in the transfer of siRNAs into the RISC (Liu et al., 2003). Generation of siRNAs from dsRNA in Drosophila embryo extracts, unwinding of the siRNA duplex, and incorporation into the RISC require ATP (Nykanen et al., 2001). In contrast, human Dicer does not seem to require ATP for processing of dsRNA into siRNA molecules (Zhang et al., 2002).

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[3.] Ww/Fragment 011 25 - Diskussion
Zuletzt bearbeitet: 2014-10-29 07:45:40 Hindemith
Fragment, Gesichtet, Hammond 2005, KomplettPlagiat, SMWFragment, Schutzlevel sysop, Ww

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Untersuchte Arbeit:
Seite: 11, Zeilen: 25-30
Quelle: Hammond 2005
Seite(n): 5825, Zeilen: r.col. 14 ff.
Chromatographic purification of RISC nuclease activity from Drosophila cells revealed several RISC components. The first identified component was Argonaute2 (Ago2) (Hammond et al, 2001). This protein is a member of a gene family conserved in most eukaryotic and several prokaryotic genomes. The C. elegans homolog, rde-1, was previously identified in a genetic screen for RNAi-deficient mutants, reinforcing its connection with RNAi (Tabara et al, [1999).] Chromatographic purification of RISC nuclease activity from Drosophila cells revealed several RISC components. The first identified component was Argonaute2 (Ago2) [43]. This protein is a member of a gene family conserved in most eukaryotic and several prokaryotic genomes. The C. elegans homolog, rde-1, was previously identified in a genetic screen for RNAi-deficient mutants, reinforcing its connection with RNAi [12].

[12] Tabara, H., Sarkissian, M., Kelly, W.G., Fleenor, J., Grishok, A., Timmons, L., Fire, A. and Mello, C.C. (1999) The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99, 123–132.

[43] Hammond, S.M., Boettcher, S., Caudy, A.A., Kobayashi, R. and Hannon, G.J. (2001) Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293, 1146–1150

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Sichter
(SleepyHollow02), Hindemith



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Letzte Bearbeitung dieser Seite: durch Benutzer:Hindemith, Zeitstempel: 20141029171041