Transcript Recovery and analysis of old/ancient DNA: molecular archaeology

Double-stranded RNA-induced RNA interference causes destruction
of a specific mRNA in C. elegans
uninjected, no probe
uninjected, mex-3 probe
antisense mex-3 RNA,
mex-3 probe
double-stranded mex-3 RNA
injected, mex-3 probe
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)
Mechanism of RNAi post-transcriptional gene silencing
dsRNA
5’
small interfering RNAs
5’
(inactive, 250-500kDa complex)
(a critical step in the activation of RISC)
RNA-induced silencing complex (active, 100kDa complex)
(endonucleolytic cleavage in the region of homology)
Zamore, P. D. Science 296, 1265-1269 (2002)
Dicer
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 Dicer
is 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)
RISC
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 unwound
siRNA as a guide to substrate
selection
Hannon, G. J. Nature 418, 244-251 (2002)
Unanswered question
•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
RNAi can silence gene expression through other mechanisms:
Histone methylation occurs at loci homologous to the siRNA target
histone
methyltransferase
RNA-dependent
RNA 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
Synthesis of double-stranded RNA (dsRNA) in vitro
cut A
cut B
cut A, SP6 polymerase, NTPs
cut A
SP6
T7
plasmid
3’
5’
cut B, T7 polymerase, NTPs
SP6
T7
5’
complementary 21-mer
RNA oligonucleotides
5’-GGCAAGGUCCAGCUGAACU-3’
|||||||||||||||||
3’-UACCGUUCCAGGUCGACUU-5’
anneal
5’
3’
3’
5’
800-1200 bp dsRNA
cut B
3’
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
Agrobacteriu
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
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
Terminator technology
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
How to produce sterile seeds on demand:
Ribosome inactivating protein (RIP) under the control of
a late embryonic development induced promoter,
remove transcription terminator by Cre/loxP system
Plants produce viable seeds
x
stop
RIP
loxP
loxP
RIP
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 Syngenta
Embarrassment (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
population
c. Will the plant harm beneficial animals and insects?
d. How quickly will resistant strains of pathogens arise?
2. Human health
is 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