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#1

methaangas

    methaangas


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Geplaatst op 28 oktober 2004 - 17:48

Hallo,


Ik moet voor werkcollege een college geven over RNAi, ik heb al verscheidene bedrijven en sites geraadpleegd. Ik heb nu een enorme stapel info, maar er wordt telkens weinig of niets verteld over de ontdekking van RNAi.

Dus: als iemand een goed artikel of een goede site kent, gelieve dat dan hier te posten.


Greets

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#2


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Geplaatst op 28 oktober 2004 - 18:14

Scientific American Censors of the Genome; August 2003

A Strange Silence
THE FIRST HINTS of the RNAi phenomenon surfaced 13 years
ago. Richard A. Jorgensen, now at the University of Arizona,
and, independently, Joseph Mol of the Free University of Amsterdam
inserted into purple-flowered petunias additional copies
of their native pigment gene. They were expecting the engineered
plants to grow flowers that were even more vibrantly violet. But
instead they obtained blooms having patches of white.
Jorgensen and Mol concluded that the extra copies were
somehow triggering censorship of the purple pigment genes—
including those natural to the petunias—resulting in variegated
or even albino-like flowers. This dual censorship of an inserted
gene and its native counterpart, called co-suppression, was later
seen in fungi, fruit flies and other organisms.
Clues to the mystery of how genes were being silenced came
a few years later from William G. Dougherty’s lab at Oregon
State University. Dougherty and his colleagues started with tobacco
plants that had been engineered to contain within their
DNA several copies of the CP (coat protein) gene from tobacco
etch virus. When these plants were exposed to the virus, some
of the plants proved immune to infection. Dougherty proposed
that this immunity arose through co-suppression. The plants apparently
reacted to the initial expression of their foreign CP
genes by shutting down this expression and subsequently also
blocking expression of the CP gene of the invading virus (which
needed the coat protein to produce an infection). Dougherty’s
lab went on to show that the immunity did not require synthesis
of the coat protein by the plants; something about the RNA
transcribed from the CP gene accounted for the plants’ resistance
to infection.
The group also showed that not only could plants shut off
specific genes in viruses, viruses could trigger the silencing of selected
genes. Some of Dougherty’s plants did not suppress their
CP genes on their own and became infected by the virus, which
replicated happily in the plant cells. When the researchers later
measured the RNA being produced from the CP genes of the affected
plants, they saw that these messages had nearly vanished—
infection had led to the CP genes’ inactivation.
Meanwhile biologists experimenting with the nematode
Caenorhabditis elegans, a tiny, transparent worm, were puzzling
over their attempts to use “antisense” RNA to inactivate the
genes they were studying. Antisense RNA is designed to pair up
with a particular messenger RNA sequence in the same way that
two complementary strands of DNA mesh to form a double helix.
Each strand in DNA or RNA is a chain of nucleotides, genetic
building blocks represented by the letters A, C, G and either
U (in RNA) or T (in DNA). C nucleotides link up with Gs,
and As pair with Us or Ts. A strand of antisense RNA binds to
a complementary messenger RNA strand to form a doublestranded
structure that cannot be translated into a useful protein.
Over the years, antisense experiments in various organisms
have had only spotty success. In worms, injecting antisense RNAs
seemed to work. To everyone’s bewilderment, however, “sense”
RNA also blocked gene expression. Sense RNA has the same sequence
as the target messenger RNA and is therefore unable to
lock up the messenger RNA within a double helix.
The stage was now set for the eureka experiment, performed
five years ago in the labs of Andrew Z. Fire of the Carnegie Institution
of Washington and Craig C. Mello of the University of
Massachusetts Medical School. Fire and Mello guessed that the
previous preparations of antisense and sense RNAs that were
being injected into worms were not totally pure. Both mixtures
probably contained trace amounts of double-stranded RNA. They suspected that the double-stranded RNA was alerting the censors.
To test their idea, Fire, Mello and their colleagues inoculated
nematodes with either single- or double-stranded RNAs that
corresponded to the gene unc-22, which is important for muscle
function. Relatively large amounts of single-stranded unc-22
RNA, whether sense or antisense, had little effect on the nematodes.
But surprisingly few molecules of double-stranded unc-22
RNA caused the worms—and even the worms’ offspring—to
twitch uncontrollably, an unmistakable sign that something had
started interfering with unc-22 gene expression. Fire and Mello
observed the same amazingly potent silencing effect on nearly
every gene they targeted, from muscle genes to fertility and viability
genes. They dubbed the phenomenon “RNA interference”
to convey the key role of double-stranded RNA in initiating censorship
of the corresponding gene.
Investigators studying plants and fungi were also closing
in on double-stranded RNA as the trigger for silencing. They
showed that RNA strands that could fold back on themselves
to form long stretches of double-stranded RNA were potent inducers
of silencing. And other analyses revealed that a gene that
enables cells to convert single-stranded RNA into doublestranded
RNA was needed for co-suppression. These findings
suggested that Jorgensen and Mol’s petunias recognized the extra
pigment genes as unusual (through a mechanism that is still
mysterious) and converted their messenger RNAs into doublestranded
RNA, which then triggered the silencing of both the
extra and native genes. The concept of a double-stranded RNA
trigger also explains why viral infection muzzled the CP genes
in Dougherty’s plants. The tobacco etch virus had created double-
stranded RNA of its entire viral genome as it reproduced,
as happens with many viruses. The plant cells responded by cutting
off the RNA messages of all genes associated with the virus,
including the CP genes incorporated into the plant DNA.
Biologists were stunned that such a powerful and ubiquitous
system for regulating gene expression had escaped their notice
for so long. Now that the shroud had been lifted on the phenomenon,
scientists were anxious to analyze its mechanism of
action and put it to gainful employment.
Slicing and Dicing Genetic Messages
RNA INTERFERENCE was soon observed in algae, flatworms
and fruit flies—diverse branches of the evolutionary tree.
Demonstrating RNAi within typical cells of humans and other
mammals was considerably trickier, however.
When a human cell is infected by viruses that make long double-
stranded RNAs, it can slam into lockdown mode: an enzyme
known as PKR blocks translation of all messenger RNAs—both normal and viral—and the enzyme RNAse L indiscriminately
destroys the messenger RNAs. These responses to doublestranded
RNA are considered components of the so-called interferon
response because they are triggered more readily after
the cells have been exposed to interferons, molecules that infected
cells secrete to signal danger to neighboring cells.
Unfortunately, when researchers put artificial double-stranded
RNAs (like those used to induce RNA interference in worms
and flies) into the cells of mature mammals, the interferon response
indiscriminately shuts down every gene in the cell. A
deeper understanding of how RNA interference works was
needed before it could be used routinely without setting off the
interferon alarms. In addition to the pioneering researchers already
mentioned, Thomas Tuschl of the Rockefeller University,
Phillip D. Zamore of the University of Massachusetts Medical
School, Gregory Hannon of Cold Spring Harbor Laboratory
in New York State and many others have added to our current
understanding of the RNA interference mechanism. of interest to ascertain the gene’s function. Now the dream of
easily silencing a single, selected gene in mammalian cells was
suddenly attainable. With siRNAs, almost any gene of interest
can be turned off in mammalian cell cultures—including human
cell lines—within a matter of hours. And the effect persists for
days, long enough to complete an experiment.
A Dream Tool
AS HELPFUL AS RNA interference has become to mammal biologists,
it is even more useful at the moment to those who study
lower organisms. A particular bonus for those studying worms
and plants is that in these organisms the censorship effect is amplified
and spread far from the site where the double-stranded
RNA was introduced. This systemic phenomenon has allowed
biologists to exploit RNAi in worms simply by feeding them bacteria
engineered to make double-stranded RNA corresponding
to the gene that should be shut down.
Because RNA interference is so easy to induce and yet so
powerful, scientists are thinking big. Now that complete genomes—
all the genes in the DNA—have been sequenced for a
variety of organisms, scientists can use RNA interference to explore
systematically what each gene does by turning it off. Recently
four groups did just that in thousands of parallel experiments,
each disabling a different gene of C. elegans. A similar
genome-wide study is under way in plants, and several consortia
are planning large RNAi studies of mammalian cells.
RNA interference is being used by pharmaceutical companies
as well. Some drug designers are exploiting the effect as a
shortcut to screen all genes of a certain kind in search of promising
targets for new medicines. For instance, the systematic silencing
of genes using RNAi could allow scientists to find a gene
that is critical for the growth of certain cancer cells but not so
important for the growth of normal cells. They could then develop
a drug candidate that interferes with the protein product
of this gene and then test the compound against cancer. Biotech
firms have also been founded on the bet that gene silencing by RNAi could itself become a viable therapy to treat cancer, viral
infections, certain dominant genetic disorders and other diseases
that could be controlled by preventing selected genes from
giving rise to illness-causing proteins.
Numerous reports have hinted at the promise of siRNAs for
therapy. At least six labs have temporarily stopped viruses—
HIV, polio and hepatitis C among them—from proliferating in
human cell cultures. In each case, the scientists exposed the cells
to siRNAs that prompted cells to shut down production of proteins
crucial to the pathogens’ reproduction. More recently,
groups led by Judy Lieberman of Harvard Medical School and
Mark A. Kay of the Stanford University School of Medicine
have reported that siRNAs injected under extremely high pressure
into mice slowed hepatitis and rescued many of the animals
from liver disease that otherwise would have killed them.
Despite these laboratory successes, it will be years before
RNAi-based therapies can be used in hospitals. The most diffi-
cult challenge will probably be delivery. Although the RNAi effect
can spread throughout a plant or worm, such spreading
does not seem to occur in humans and other mammals. Also,
siRNAs are very large compared with typical drugs and cannot
be taken as pills, because the digestive tract will destroy them
rather then absorb them. Researchers are testing various ways
to disseminate siRNAs to many organs and to guide them
through cells’ outer membranes. But it is not yet clear whether
any of the current strategies will work.
Another approach for solving the delivery problem is gene
therapy. A novel gene that produces a particular siRNA might
be loaded into a benign virus that will then bring the gene into
the cells it infects. Beverly Davidson’s group at the University
of Iowa, for example, has used a modified adenovirus to deliver
genes that produce siRNAs to the brain and liver of mice.
Gene therapy in humans faces technical and regulatory diffi-
culties, however.
Regardless of concerns about delivery, RNAi approaches
have generated an excitement not currently seen for antisense
and catalytic RNA techniques—other methods that, in principle,
could treat disease by impeding harmful messenger RNAs.
This excitement stems in part from the realization that RNA interference
harnesses natural gene-censoring machinery that
evolution has perfected over time.
Why Cells Have Censors
INDEED, THE GENE-CENSORING mechanism is thought to
have emerged about a billion years ago to protect some common
ancestor to plants, animals and fungi against viruses and
mobile genetic elements. Supporting this idea, the groups of
Ronald H. A. Plasterk at the Netherlands Cancer Institute and
of Hervé Vaucheret at the French National Institute of Agricultural
Research have shown that modern worms rely on RNA
interference for protection against mobile genetic elements and
that plants need it as a defense against viruses.
Yet RNA interference seems to play other biological roles as
well. Mutant worms and weeds having an impaired Dicer enzyme
or too little of it suffer from numerous developmental defects
and cannot reproduce. Why should a Dicer deficiency cause
animals and plants to look misshapen?
One hypothesis is that once nature developed such an effective
mechanism for silencing the subversive genes in viruses and
mobile DNA sequences, it started borrowing tools from the
RNAi tool chest and using them for different purposes. Each cell
has the same set of genes—what makes them different from one
another is which genes are expressed and which ones are not.
Most plants and animals start as a single embryonic cell that divides
and eventually gives rise to a multitude of cells of various
types. For this to occur, many of the genes expressed in the embryonic
cells need to be turned off as the organ matures. Other
genes that are off need to be turned on. When the RNAi machinery
is not defending against attack, it apparently pitches in
to help silence normal cellular genes during developmental transitions
needed to form disparate cell types, such as neurons and
muscle cells, or different organs, such as the brain and heart.
What then motivates the RNAi machinery to hush particular
normal genes within the cell? In some cases, a cell may nat- urally produce long double-stranded RNA specifically for this
purpose. But frequently the triggers are “microRNAs”—small
RNA fragments that resemble siRNAs but differ in origin. Whereas
siRNAs come from the same types of genes or genomic regions
that ultimately become silenced, microRNAs come from genes
whose sole mission is to produce these tiny regulatory RNAs.
The RNA molecule initially transcribed from a microRNA
gene—the microRNA precursor—folds back on itself, forming
a structure that resembles an old-fashioned hairpin. With the
help of Dicer, the middle section is chopped out of the hairpin,
and the resulting piece typically behaves very much like an
siRNA—with the important exception that it does not censor
a gene with any resemblance to the one that produced it but instead
censors some other gene altogether.
As with the RNAi phenomenon in general, it has taken biologists
time to appreciate the potential of microRNAs for regulating
gene expression. Until recently, scientists knew of only two
microRNAs, called lin-4 RNA and let-7 RNA, discovered by the
groups of Victor Ambros of Dartmouth Medical School and
Gary Ruvkun of Harvard Medical School. In the past two years
we, Tuschl, Ambros and others have discovered hundreds of additional
microRNA genes in worms, flies, plants and humans.
With Christopher Burge at M.I.T., we have estimated that
humans have between 200 and 255 microRNA genes—nearly
1 percent of the total number of human genes. The microRNA
genes had escaped detection because the computer programs designed
to sift through the reams of genomic sequence data had
not been trained to find this unusual type of gene, whose final
product is an RNA rather than a protein.
Some microRNAs, particularly those in plants, guide the slicing
of their mRNA targets, as was shown by James C. Carrington
of Oregon State University and Zamore. We and Bonnie
Bartel of Rice University have noted that plant microRNAs take
aim primarily at genes important for development. By clearing
their messages from certain cells during development, RNAi
could help the cells mature into the correct type and form the
proper structures.
Interestingly, the lin-4 and let-7 RNAs, first discovered in
worms because of their crucial role in pacing development, can
employ a second tactic as well. The messenger RNAs targeted
by these microRNAs are only approximately complementary to
the microRNAs, and these messages are not cleaved. Some other
mechanism blocks translation of the messenger RNAs into
productive proteins.
Faced with these different silencing mechanisms, biologists
are keeping open minds about the roles of small RNAs and the
RNAi machinery. Mounting evidence indicates that siRNAs not
only capture messenger RNAs for destruction but can also direct
the silencing of DNA—in the most extreme case, by literally editing
genes right out of the genome. In most cases, however, the
silenced DNA is not destroyed; instead it is more tightly packed
so that it cannot be transcribed.
From its humble beginnings in white flowers and deformed
worms, our understanding of RNA interference has come a long
way. Almost all facets of biology, biomedicine and bioengineering
are being touched by RNAi, as the gene-silencing technique
spreads to more labs and experimental organisms.
Still, RNAi poses many fascinating questions. What is the
span of biological processes that RNA interference, siRNAs and
microRNAs influence? How does the RNAi molecular machinery
operate at the level of atoms and chemical bonds? Do any
diseases result from defects in the RNAi process and in micro-
RNAs? As these questions yield to science, our understanding
of the phenomenon will gradually solidify—perhaps into a
foundation for an entirely new pillar of genetic medicine.
RNAi appears to work like this: Inside a cell, doublestranded
RNA encounters an enzyme dubbed Dicer. Using the
chemical process of hydrolysis, Dicer cleaves the long RNA into
pieces, known as short (or small) interfering RNAs, or siRNAs.
Each siRNA is about 22 nucleotides long.
Dicer cuts through both strands of the long double-stranded
RNA at slightly staggered positions so that each resulting
siRNA has two overhanging nucleotides on one strand at eitherend [see box above]. The siRNA duplex is then unwound, and
one strand of the duplex is loaded into an assembly of proteins
to form the RNA-induced silencing complex (RISC).
Within the silencing complex, the siRNA molecule is positioned
so that messenger RNAs can bump into it. The RISC will
encounter thousands of different messenger RNAs that are in
a typical cell at any given moment. But the siRNA of the RISC
will adhere well only to a messenger RNA that closely complements
its own nucleotide sequence. So, unlike the interferon
response, the silencing complex is highly selective in choosing
its target messenger RNAs.
When a matched messenger RNA finally docks onto the
siRNA, an enzyme known as Slicer cuts the captured messenger
RNA strand in two. The RISC then releases the two messenger
RNA pieces (now rendered incapable of directing protein
synthesis) and moves on. The RISC itself stays intact, free
to find and cleave another messenger RNA. In this way, the
RNAi censor uses bits of the double-stranded RNA as a blacklist
to identify and mute corresponding messenger RNAs.
David C. Baulcombe and his co-workers at the Sainsbury
Laboratory in Norwich, England, were the first to spot siRNAs,
in plants. Tuschl’s group later isolated them from fruit fly embryos
and demonstrated their role in gene silencing by synthesizing
artificial siRNAs and using them to direct the destruction
of messenger RNA targets. When that succeeded, Tuschl wondered
whether these short snippets of RNA might slip under the
radar of mammalian cells without setting off the interferon response,
which generally ignores double-stranded RNAs that are
shorter than 30 nucleotide pairs. He and his co-workers put synthetic
siRNAs into cultured mammalian cells, and the experiment
went just as they expected. The target genes were silenced;
the interferon response never occurred.
Tuschl’s findings rocked the biomedical community. Geneticists
had long been able to introduce a new gene into mammalian
cells by, for example, using viruses to ferry the gene into
cells. But it would take labs months of labor to knock out a gene

#3

methaangas

    methaangas


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Geplaatst op 31 oktober 2004 - 15:11

Thanks!

Maar heb wel een aantal vraagjes
'additional copies of their native pigment gene' Kopieën van de pigmentgenen in vitro gemaakt en dan later ingespoten of via klonen of ??

CP gene?? Simpel: Wat is het Coat Proteïn gene?

gene from tobacco ETCH virus -> ??

Maar ga de vraag wel beperken tot de petunia's en de tabakplanten

#4

Bas

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Geplaatst op 31 oktober 2004 - 15:38

'additional copies of their native pigment gene' Kopieën van de pigmentgenen in vitro gemaakt en dan later ingespoten of via klonen of ??

Ingebracht in het DNA van de plant via genetische modificatie (dus via kloneren zou je kunnen zeggen).

CP gene?? Simpel: Wat is het Coat Proteïn gene?

Het gen dat codeert voor de eiwitten van de virusmantel (het omhulsel).

gene from tobacco ETCH virus -> ??

Dit is gewoon een virussoort.
Ik wou dat ik een elektron was, dan kon ik altijd paren

#5

methaangas

    methaangas


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Geplaatst op 31 oktober 2004 - 16:23

Thanks Bas
The plants apparently reacted to the initial expression of their foreign CP
genes by shutting down this expression and subsequently also
blocking expression of the CP gene of the invading virus.


De planten bleken te reageren met de eerste expressie van hun vreemde CP genen door de expressie te beeindigen en vervolgens ook de expressie van het CP gen van het indringende gen te blokkeren

Is deze zin juist vertaald, want versta het toch niet helemaal :shock:

#6

Bas

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Geplaatst op 31 oktober 2004 - 17:22

The plants apparently reacted to the initial expression of their foreign CP genes by shutting down this expression and subsequently also blocking expression of the CP gene of the invading virus.

Enigszins vrijelijk vertaald: Blijkbaar reageerden de planten op de expressie van hun ingebrachte CP-genen door deze expressie stop te zetten en vervolgens ook de expressie te blokkeren van het CP-gen van het binnendringende virus.
Dus als er een virus binnendringt met dezelfde CP-genen dan zullen ook deze virusgenen niet meer worden vertaald in eiwit. RNAi is dus waarschijnlijk van nature een afweermechanisme van de plant tegen virussen.
Ik wou dat ik een elektron was, dan kon ik altijd paren

#7

methaangas

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Geplaatst op 31 oktober 2004 - 20:51

Nogmaals thanks :shock:





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