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Technologii inzerční mutageneze lze využít i u živočichů. Zda se využívají
např. transpozony odvozené z Drosophily (transpozon Minos, viz schéma
vlevo nahoře (Klinakis et al., 2000). V tomto případě bylo nutne provést
kotransfekci s tzv. helper plasmiem, kódujícím transponázu (neautonomní
transpozon). Neo kóduje rezistenci k neomycinu, šipky ukazují směr
transkripce řízený přislušnými promotory, pA je polyadenylační signál, ori
je počátek replikace viru SV40, S-P je promotor téhož viru. Pro identifikaci
inzercí „in frame“ se zasaženými geny lze využít transpozony, obsahující
fůzi akceptorových míst sestřihu s ORF reportérového genu, např. lacZ-neo
(bez AUG kodonu). Tento přístup umožňuje identifikovat inzerce do
aktivních genů prostřednictvím selekce inzerčních mutantů na rezistenci k
neomycinu, resp. vykazující β-galaktozidázovou aktivitu (Klinakis et al.,
2000).
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It has been found that dsRNA might be either an intermediate or a trigger in PTGS.
In the first case, dsRNA is formed by the action of RNA-dependent RNA polymerases (RdRPs), which use specific transcripts as a template. It is still not
clear, how these transcripts are recognized, but it might be e.g. abundant RNA that is a result of viral amplification or transcription of foreign DNA.
It is not clear, how the foreign DNA might be recognized, possibly, lack of bound proteins on the foreign “naked” DNA and its subsequent “signature”
(e.g. by specific methylation pattern) during packing of the foreign DNA into the chromatin structure might be involved.
The highly abundant transcripts might be recruited to the RdRPs by the defects in the RNA processing, e.g. lack of polyadenylation.
In the case when dsRNA is a direct trigger, there are two major RNA molecules involved in the process: Short interference RNA (siRNA) and micro RNA
(miRNA), both encoded by the endogenous DNA.
These two functionally similar molecules differ in their origin:
siRNAs are dominantly product of the cleavage of the long dsRNA that are produced by the action of cellular or viral RdRPs. However, there are also
endogenous genes, e.g. short hairpin RNAs (shRNAs) allowing production of the siRNA (see the figure).
miRNAs are involved in the developmental-specific regulations and are product of transcription of endogenous genes encoding for small dsRNAs with
specific structure (see the figure).
In addition to siRNAs, there are trans-acting siRNAs (tasiRNAs) that are a special class of siRNAs that appear to function in development (much like
miRNAs) but have a unique mode of origin involving components of both miRNA and siRNA pathways.
Developmental regulations via miRNAs are more often used in animals then in plants.
The dsRNAs of all origins and pre miRNAs are cleaved by DICER or DICER-like (DCL) enzyme complexes with RNAse activity, leading to production of
siRNAs and miRNA, respectively.
These small RNAs are of 21-24 bp long and bind either to RNA-induced transcriptional silencing complex (RITS) or RNA-induced silencing
komplex (RISC).
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In siRNA and miRNA biogenesis, DICER or DICER-like (DCL) proteins
cleave long dsRNA or foldback (hairpin) RNA into ~ 21 – 25 nt fragments.
Dicer’s structure allows it to measure the RNA it is cleaving. Like a cook
who “dices” a carrot, DICER chops RNA into uniformly-sized pieces.
Note the two strands of the RNA molecule. The cleavage sites are indicated by
yellow arrows.
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ARGONAUTE proteins bind small RNAs and their targets and it is an
important part of both RITS and RISC complexes.
ARGONAUTE proteins are named after the argonaute1 mutant of
Arabidopsis; ago1 has thin radial leaves and was named for the octopus
Argonauta which it resembles (see the figure).
ARGONAUTE proteins were originally described as being important for plant
development and for germline stem-cell division in Drosophila melanogaster.
ARGONAUTE proteins are classified into three paralogous groups: Argonautelike
proteins, which are similar to Arabidopsis thaliana AGO1; Piwi-like proteins,
which are closely related to D. melanogaster PIWI (P-element induced wimpy
testis); and the recently identified Caenorhabditis elegans-specific group 3
Argonautes.
Members of a new family of proteins that are involved in RNA silencing mediated
by Argonaute-like and Piwi-like proteins are present in bacteria, archaea and
eukaryotes, which implies that both groups of proteins have an ancient origin.
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The number of Argonaute genes that are present in different species varies. There
are 8 Argonaute genes in humans (4 Argonaute-like and 4 Piwi-like), 5 in the D.
melanogaster genome (2 Argonaute-like and 3 Piwi-like), 10 Argonaute-like in A.
thaliana, only 1 Argonaute-like in Schizosaccharomyces pombe and at least 26
Argonaute genes in C. elegans (5 Argonaute-like, 3 Piwi-like and 18 group 3
Argonautes).
http://youdpreferanargonaute.com/2009/06/
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MicroRNAs are encoded by MIR genes, fold into hairpin structures that are
recognized and cleaved by DCL (Dicer-like) proteins.
In summary, siRNAs-mediates silencing via post-transcriptional and
transcriptional gene silencing, while miRNAs -mediate slicing of mRNA
and translational repression.
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In 2006, Andrwe Z. Fire and Craig C. Mello were honored by the Nobel prize “for
their discovery of RNA interference - gene silencing by double-stranded
RNA“.
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