lectut btn-202-ppt-l37. transgene silencing
TRANSCRIPT
Transgene Silencing
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Lecture- 37
• Gene silencing is a general term describing epigenetic process of gene regulation. The term gene silencing is generally used to describe the “switching off” of a gene by a mechanism other than mutation. That is , a gene which would be expressed under normal circumstances is switched off by machinery in the cell.
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• Genes are regulated at either the transcriptional or post-transcriptional level.
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Phenomena of gene silencing was
first observed in Petunia
An attempt was made to overexpress chalone synthase (anthrocyanin pigment
gene) in Petunia to darken flower color by introducing another copy of chalone
synthase gene (transgene)
The result was the loss of pigment.
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Co-suppression:
Suppression of the expression of
both endogenous gene and
transgene.
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Two mechanisms can explain this transgene-
mediated gene silencing
•Transcriptional gene silencing(TGS)
•Post Transcriptional Gene Silencing
(PTGS)
mRNA is made, but then degraded
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• Transcriptional gene silencing is the result of histone modifications, creating an environment of heterochromatin around a gene that makes it inaccessible to transcriptional machinery (RNA polymerase, transcription factors, etc. ).
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• Post-transcriptional gene silencing is the result of mRNA of a particular gene being destroyed or blocked. The destruction of the mRNA prevents translation to form an active gene product (in most cases, a protein). A common mechanism of post-transcriptional gene silencing is RNA interference (RNAi).
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Other names of post-transcriptional gene
silencing (PTGS) :
– RNA silencing
•– RNA interference (RNAi)
•– In certain fungi: quelling
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Before RNAi was well characterized, it was called by
other names, including post transcriptional gene
silencing (PTGS) and transgene silencing. Only after
these phenomena were characterized at the molecular
level it was observed that they were the same
phenomena.
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History of post-transcriptional gene silencing
Definition: the ability of exogenous double-stranded
RNA (dsRNA) to suppress the expression of the gene
which corresponds to the dsRNA sequence.
•1990 Jorgensen
Introduction of transgenes homologous to
endogenous genes often resulted in plants with both
genes suppressed.
Called Co-suppression
Resulted in degradation of the endogenous and the
transgene mRNA11
•1995 Guo and Kemphues
Injection of either antisense or sense RNAs in the
germline of C. elegans was equally effective at
silencing homologous target genes
•1998 Mello and Fire
Extension of above experiments, combination of
sense and antisense RNA (dsRNA) was 10 times
more effective than single strand RNA
Contd….
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Experiment demonstrating RNAi (conducted by Mello and Fire in 1998)
Phenotypic effect after injection of single-stranded or double-stranded unc-22 RNA into the gonad of C. elegans. The unc-22 gene encodes a myofilament protein. Decrease in unc-22 activity is known to produce severe twitching movements. Injected double-stranded RNA, but not single-stranded RNA, induced the twitching phenotype in the progeny. 13
The Nobel Prize in Physiology or Medicine 2006 was awarded jointly to Andrew Z. Fire and Craig C.
Mello "for their discovery of RNA interference - gene silencing by double-stranded RNA"
Andrew Z. Fire Craig C. Mello 14
What is RNA interference /PTGS?
• dsRNA needs to be directed against an exon, not an
intron in order to be effective
• homology of the dsRNA and the target gene/mRNA is
required
• targeted mRNA is lost (degraded) after RNAi
• the effect is non-stoichiometric; small amounts of
dsRNA can wipe out an excess of mRNA (pointing to
an enzymatic mechanism)
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• Double-stranded RNA triggers cleavage of homologous
mRNA
• The life cycle and replication of many RNA virusesinvolves a double-stranded RNA stage, so it is likelythat part of the RNA interference machinery evolved as a defense against these viruses.
• PTGS-defective plants are more sensitive to infection by RNA viruses
• In RNAi defective nematodes, transposons are much more active
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Double-stranded RNAs are produced by:
– transcription of inverted repeats
– viral replication
– transcription of RNA by RNA-dependent RNA-
polymerases (RdRP)
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RNAi can be induced
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RNA interference process and the biochemical machinery involved
ds RNA is cut into short double-stranded fragments called small interfering RNAs (siRNAs) by Dicer, a ribonuclease III-like nuclease .
siRNAs are then separated into single strands and integrated into an active RNA-induced silencing complex (RISC)
The antisense strand is loaded into RISC and links the complex to the mRNA strand by base-pairing.
The RISC complex cuts the mRNA strand, and the mRNA is subsequently degraded.
• Exogenous, e.g. a virus with an RNA genome • Endogenous, e.g. Stem-loop structure of pre-microRNAs
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