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gene silencing
gene or
genes usually from an unrelated organism;
such genes are called TRANSGENES.
And the plants containing transgenes are called as
TRANSGENIC PLANTS.
2. Truncation of T-DNA
Sometimes the transgene introduced is not in its
proper sequence or structure which leads to
production of Truncated protein.
Thus improper expression of transgene.
4.Effect of ploidy
Even number of copies of introduced transgene show much
better expression as compared to odd number of copies.
This occurs at transcriptional level may be because of
direct physical association or pairing of alleles.
Reduced gene expression is observed in triploids as
compared to diploids.
5. Integration sites
The surrounding DNA sequences like promoters,
enhancers, silencers and secondary structures play a vital
role in determing the expression level of the transgene
introduced.
IV.
V.
Transcriptional silencing
Post-transcriptional silencing
The mechanism is as co suppression
Co-suppression is inhibition of an endogenous gene by the
presence of a homologous sense transgene.
It was seen that when experiments designed to increase the
levels of an endogenous protein by introducing extra copies
of the corresponding gene.
Co-suppression is a systemic phenomena
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Example:
To pigmentation in petunia
Promoters silenced
Promoters active
Genes hypermethylated Gene hypermethylated
in coding region
in promoter region
It is systemic silencing
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Types
of
post-transcriptional
silencing (PTGS) :
gene
1. Antisense technology
2. Ribozyme technology
3. RNA interference
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Ribozyme technology
Ribozyme are catalytic RNA molecules that destroy
targeted mRNA by site-specific cleavage
They are recycled after the cleavage reaction and can
therefore inactivate many mRNA molecules
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RNA interference
ds RNA needs to be directed against an exon, not an
intron in order to be effective
Homology of the ds RNA and the target gene/mRNA is
required
Targeted mRNA is lost (degraded)
The effect is non- stoichiometric; small amounts of
ds RNA can wipe out an excess of mRNA (pointing to
an enzymatic mechanism)
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Dicer
Double-stranded RNA processed into si RNAs
by enzyme RNAseIII, specifically the Dicer family
Processive enzyme - no larger intermediates.
Dicer family proteins are ATP-dependent nucleases.
These proteins contain an amino-terminal Helicase
domain, dual RNAseIII domains in the carboxyterminal segment, and ds RNA-binding motifs.
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RISC complex
RISC is a large (~500-kDa) RNA- multiprotein complex, which
triggers mRNA degradation in response to si RNA
some components have been defined by genetics, but function
is unknown, e.g.
unwinding of double-stranded si RNA (Helicase )
ribonuclease component cleaves mRNA (Nuclease )
amplification of silencing signal (RNA-dependent RNA
polymerase )
cleaved mRNA is degraded by cellular exonucleases
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Different
molecules
classes
of
small
RNA
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si RNAs
Small interfering RNAs that have an integral role in
the phenomenon of RNA interference(RNAi),
a form of post-transcriptional gene silencing
RNAi: 21-25 nt fragments, which bind to the
complementary portion of the target mRNA
and tag it for degradation
A single base pair difference between the si RNA
template and the target mRNA is enough to block
the process.
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mi RNAs
micro/small temporal RNAs
derive from ~70 nt ss RNA (single-stranded RNA),
which forms a stem-loop; processed to 22nt RNAs
Found in:
Drosophila, C. elegans, HeLa cells
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