uninjected, mex-3 probeuninjected, no probe
antisense mex-3 RNA, mex-3 probe
double-stranded mex-3 RNA injected, mex-3 probe
Double-stranded RNA-induced RNA interference causes destruction of a specific mRNA in C. elegans
Guo, S. and Kemphues, K. J. Cell 81, 611-620 (1995)Fire, A. et al. Nature 391, 809 (1998)
Key points of C. elegans experiment
•substoichiometric amounts of dsRNA relative to the targeted mRNA are required to completely eliminate the mRNA (i.e. the dsRNA is catalytic)
•dsRNA is 10-100X better than antisense or sense RNA
•doesn’t work if introns or promoters are targeted by the dsRNA
•doesn’t interfere with transcription initiation or elongation (it is possible to target a single gene in an operon) (i.e. RNAi is a post-transcriptional phenomena)
•the targeted mRNA is degraded (i.e. it can’t be detected by probes)
•dsRNA can cross cellular boundaries (i.e. there is a transport mechanism)
dsRNA
5’
5’
RNA-induced silencing complex (active, 100kDa complex)
small interfering RNAs
Mechanism of RNAi post-transcriptional gene silencing
(inactive, 250-500kDa complex)
(endonucleolytic cleavage in the region of homology)
(a critical step in the activation of RISC)
Zamore, P. D. Science 296, 1265-1269 (2002)
Dicer contains 5 domains• 2 catalytic RNase domains• a dsRNA-binding domain• a helicase domain• a PAZ domain
Dicer is thought to work as a dimer
One of the catalytic sites in Diceris defective
Thus, instead of cleaving from~9-11 nt, like bacterial RNase III Dicer cleaves ~22 nt (see panel B)
Hannon, G. J. Nature 418, 244-251 (2002)
Dicer
RISC contains at least 4 subunits•Argonaute (5 homologs in Dros.)•dFXR (the Dros. homologue of human fragile X mental retardation protein)•Vasa intronic gene (VIG)•nuclease Activated RISC uses the unwoundsiRNA as a guide to substrateselection
RISC
Hannon, G. J. Nature 418, 244-251 (2002)
•How does RNAi spread throughout and organism, even when triggered by minute quantities of dsRNA?
-Requires a system to pass a signal from one cell to another
-Requires a strategy for amplifying the signal
Unanswered question
RNAi can silence gene expression through other mechanisms:Histone methylation occurs at loci homologous to the siRNA target
histonemethyltransferase
RNA-dependentRNA polymerase
Matzke, M. and Matzke, A. J. M. Science 301, 1060-1061 (2003)Schramke, V. and Allshire, R. Science 301, 1069-1074 (2003)
Questions about RNAi
•What is the endogenous biological function of the RNAi machinery?
Gene Regulation
Protection from viruses.
It could to silence transposons
•Is RNAi under negative regulation?
how about RNases that digest siRNAs
•What are neurons refractory to RNAi?
mammals
cut A
cut A, SP6 polymerase, NTPs
SP6 T7
5’3’
cut B
cut B, T7 polymerase, NTPs
SP6 T7
5’ 3’anneal
T7
SP6
cut B
plasmid
cut A
5’ 3’
5’3’
5’-GGCAAGGUCCAGCUGAACU-3’ |||||||||||||||||3’-UACCGUUCCAGGUCGACUU-5’
complementary 21-merRNA oligonucleotides
800-1200 bp dsRNA
Synthesis of double-stranded RNA (dsRNA) in vitro
diploid and polyploid species of
Chrysanthemum
Gene transfer to plants
• Plant callus and cell culture• Agrobacterium (and Rhizobium)
mediated transformation• Direct DNA transformation• Plant virus vectors
Somatic plant cells have high developmental plasticity (in contrast to most animal cells)--it’s as if they are all embryonic stem cells
– Cells from most parts of a plant can regenerate an entire new plant (totipotent)
– Plant cells can be cultured in suspension, genetically manipulated, then used to generate transgenic plants
Plant transgenics have been used to engineer:
– Insect resistance (Bacillus thuringiensis [Bt] toxin)– Microbial pathogen resistance (e.g. overexpression of naturally-occurring plant defense genes, anti-fungal peptides “defensins”– Herbicide tolerance (glyphosate [Roundup] resistance gene)– Improved nutritional value (“golden rice” containing 1 bacterial and 2 daffodil genes for Vitamin A production).
Plant tissue culture• “explant” (tissue sample) taken,
disinfected
• With correct balance of plant hormones, a “callus” will form
• Callus transferred to liquid medium and agitated to yield a cell culture
• Single cells can be grown on plates to yield new calluses
Regeneration of plants from cell culture
• Change phytohormone levels to get differentiation– High auxin, roots develop– High cytokinin, shoots develop– In both cases plant formation can be induced
OR• Induce somatic embryogenesis: under specific
conditions cells produce embryo-like structures that can develop into fertile plants
Plant cell transformation
• Infection by Agrobacterium tumefaciens and its relatives
• Chemical transformation of protoplasts (cells lacking cell walls)
• Particle bombardment• Viral infection
Agrobacterium transformation
• Plant tumors (crown gall disease) induced by bacterium Agrobacterium tumefaciens
• Tumors induced by Ti plasmids (140-235 kb) transferred to the plant cells by bacterium (Ti, Tumor inducing plasmids)
• Part of plasmid (T-DNA, 23 kb) integrates into plant genome (randomly): confers unregulated growth (hence a tumor) and directs synthesis of “opines”
• Opines provide food (carbon and nitrogen source) for bacterium, tumor provides a home, what a deal
A typical Ti plasmid• Virulence genes are responsible for T-DNA transfer--induced following bacterial attachment to plant wound
• T-DNA is flanked by “border sequences” (25 bp imperfect direct repeats) involved in the transfer process: the right-hand border sequence is sufficient for transfer
• T-DNA is excised by Vir gene products and transferred to plant cell via a conjugative pilus
T-DNA genes
• Encode phytohormones to promote unregulated growth (oncogenes)
• These oncogenes can be deleted to “disarm” the T-DNA
• Disarmed T-DNA sequences are used for transformation
An early ‘disarmed’ Ti plasmid
Transfer could be screened for by opine synthesis (product of the nos gene)
Manipulating the Ti plasmid
• Very large--difficult to manipulate in vitro• Transgene-containing T-DNA can be
created by recombination in Agrobacterium:
T-DNA
Manipulating the Ti vector
• Alternative approach: use two plasmids– One with genes for virulence (“helper”
plasmid)– The other with T-DNA sequences--smaller,
can use classical cloning techniques
Selection for T-DNA transfer
• Drug resistance (e.g. aminoglycoside antibiotics)• Herbicide resistance (e.g. glyphosate [Roundup])
• Concern over potential harm (to health and/or environment) from these markers has driven development of other methods– manA gene: confers growth on the sugar mannose as a
sole carbon source– Use of cre-lox mediated deletions to remove markers from
transgenic plants
Agrobacterium-mediated transformation
Kanamycin: selection for T-DNA transfer
Carbenicillin: kills Agrobacterium
New developments: not just Agrobacterium can transfer genes via a T-DNA vector
Closely related bacteria (Rhizobium species) can do the same
This circumvents many patents, and researchers behind this work have made the technology freely available
www.bioforge.net, see the “Transbacter” section
Fig. 10.25. Genetically engineered rice containing a
biosynthetic pathway for beta-carotene
Direct DNA transfer to plants
• Protoplast transformation– Make protoplasts (plant cells lacking cell walls)– Addition of DNA in the presence of polyethylene
glycol– Electroporation– Transformants selected on the basis of marker
genes– Regenerate whole plants
Direct DNA transfer to plants
• Particle bombardment– Particles coated with transforming DNA,
fired through plant cells (and nuclei)– Valuable method for transforming plants
that cannot be transformed by previous methods
– Works with cell cultures, embryos, leaves, etc.
Chloroplast transformation
• Many chloroplasts in each cell (high copy number and expression of transgene)
• Chloroplasts are not transmitted by pollen (easier to contain the transgene)
• Chloroplast transgenes are not subject to position effects on transgene expression
• Target integration into chloroplasts using chloroplast homology regions
Plant viruses as vectors• Naturally transforming• High-level transgene expression• No integration into host chromosome
• Mostly used for expression of foreign proteins
• DNA viruses• RNA viruses
– Most plant viruses are RNA viruses– cDNA copies of the viral genome are used
for engineering
Protection of technology and market share:“technology protection” (Monsanto)“terminator technology” (critics)
Farmers historically save a small proportion of seeds from this year’s crop for next year’s crop
Transgenic seeds: buy them once, never buy them again because of saved seeds
Unless those transgenic plants produce sterile seeds
Terminator technology
How to produce sterile seeds on demand:
stopx
RIP
RIP
Ribosome inactivating protein (RIP) under the control of a late embryonic development induced promoter, remove transcription terminator by Cre/loxP system
loxP loxP
Plants produce viable seeds
No viable seeds(the seeds are soaked in Tet before sale)
Syngenta and the release of Bt-10
Bt-11: approved by govt. for sale to farmers
Bt-10: not approved… differs from Bt-11 in that it has a marker gene (conferring ampicillin resistance)
$375,000 fine for SyngentaEmbarrassment (damage?) to the US biotech industry
Issues with recombinant plants
How will the transgene affect:1. Ecosystem health
a. Will the plant be more invasive?b. Will the plant transgenes transmit to the native
plant populationc. Will the plant harm beneficial animals and insects?d. How quickly will resistant strains of pathogens
arise?
2. Human healthis recombinant DNA-containing plant tissue safe to
eat over the long term?
3. Economics: will the choice to not grow transgenics harm farmers?
• US govt. website on agricultural biotechnology regulations: http://www.aphis.usda.gov/brs/index.html
• The Onion and recombinant broccoli