Skip to comments.Gene Silencing
Posted on 08/10/2002 5:08:09 PM PDT by gore3000
Richard Jorgensen's idea was simple enough: make bright purple petunias by splicing into the plants an extra copy of the gene that makes purple pigment. To his astonishment, the flowers bloomed white.
That curious outcome defied genetic logic. After appearing on the cover of a prominent plant journal, the puzzling result prompted a wave of scientific inquiry. Now more than a decade later, biologists are starting to get a handle on what went wrong in Dr. Jorgensen's lab and are calling the findings an important breakthrough.
Scientists working on the petunia mystery have uncovered what's shaping up to be a critical piece of cellular machinery, a process by which plant and animal cells seem to blot out the activity of particular genes. Scientists say the discovery may help explain a lot that had perplexed them about life's basic functions, and they are already applying it as a research tool in the hunt for new medicines. Venture capitalists also are betting that it will yield super drugs that act like molecular torpedoes aimed at HIV or cancer. Scores of companies and academic labs have joined the hunt.
An experiment at the Massachusetts Institute of Technology this year has shown that HIV infection can be slowed in a lab dish by using the new science to aim RNA molecules at the genetic code of the virus. Others are under way to see if the technique can do battle with cancer in laboratory mice.
Work on the new gene-silencing phenomenon, also known as "RNE interference," is helping fill a knowledge gap exposed by the Human Genome Project. When the human genetic code was published last year, scientists realized they only knew what about 2% of our DNA was for. Equally disconcerting, about half of human DNA seemed to be not human at all, but rather a junkyard of debris left by eons of invasion by viruses and other parasites.
The latest research on gene silencing looks like it will explain some of that. In some organisms, the process appears to be acting like the genome's virus-protection software, erasing the effects of corrupted junk genes.
Last week, a Cambridge, Mass., start-up founded by scientists at MIT raised $15 million to develop treatments against hepatitis and cancer. Among the other half-dozen entrants in Nucleonics, Inc. of Malvern, Pa., a firm founded by former Wyeth immunologists who independently stumbled on the silencing effect while studying cancer in mice. German scientists have formed two companies to create gene-silencing drugs and are hoping to begin human tests within two years.
The field of biotechnology is littered with the remains of technologies that looked exciting in early laboratory tests but proved difficult to translate into treatments for humans. The RNA interference story may just be a twist on the tale of a heavily hyped technology of the early 1990's known as antisense, which has been slow to develop into usable drugs.
But for now, drug companies are using RNA interference to help locate gene targets involved in cancer and other diseases. "The wave of interest has expanded because of the reproducibility of the basic claims," says Riccardo Cortese, who studies cancer for Merck & Co. in Italy.
At Exelixis Inc. in South San Francisco, Calif., 600 people carry out large scale gene research for drug giants such as Pharmacia Corp. and Bristol-Myers Squibb Co. The company, whose labs are packed with jars of flies and worms used in such research, says nearly 80% of its gene studies now use the technology. People have adopted this like wildfire from an experimental point of view," says CEO Geoffrey Duyk. "It's a very high throughput method for turning genes off."
Dr Duyk says the technique recently yielded new drug targets for Pharmacia after the company used it to study proteins involved in Alzheimer's disease. Working with transparent worms known as C. Elegans, the company used gene silencing to methodically shut down around 10,000 worm genes, tracking the effect on the formation of a protein known to be involved in Alzheimer's in humans.
After identifying all the worm genes linked to the process, they used computer databases to find the equivalent genes in the human genome. That information was passed to Pharmacia so that it can start testing drugs.
A cell's DNA sends commands out of the nucleus in the form of RNA, a closely related molecule that is also made up of genetic code. RNA serves as the blueprint from which the body makes proteins, completing a three step relay biologists call "The Central Dogma." But the dogma can't explain everything. With gene-silencing, it's now clear there's a new class of RNA molecules whose job isn't to help make protein at all, but to stop RNA messages from doing so.
Following the petunia experiments in his lab at biotech company DNA Plant Technology Corp., Dr. Jorgensen, now at the University of ARizona, concluded that the extra copy of the pigment gene he'd added was somehow cueing the plants to shut off their purple color, sometimes only partially. One flower's pattern of purple and white looked like a man leaping. Dr. Jorgenesen named it the "Cossack Dancer."
It wasn't clear the pigment gene was shutting down, and it took another decade for someone to find the next major clue. In 1998, Andrew Fire of the Carnegie Institution, a nonprofit research laboratory in Baltimore, Md., and Craig Mello of the University of Massachusetts announced they had discovered how to design a double-stranded RNA molecule that would predictably silence any gene they chose. The effect, which they dubbed "RNA interference," appeared quite potent. Just a few molecules were enough to render a gene's activity all but undetectable in the worms they worked with.
Dr. Fire's paper set off a flurry of activity, as it dawned on scientists that if gene-silencing was working in plants, and now in worms, it might be a general phenomenon in all animals. If so, it was likely to have some deep and fundamental purpose.
At MIT, Phillip Sharp was gripped by the implications of Dr. Fire's work. Most basic mechanisms in the cell were believed to be already understood.
"It was an almost retro process," says Dr. Sharp, a Nobel Prize-winning gene researcher who also heads a new $350 million brain institute on campus. "I was just dumbfounded that it hadn't been described before."
The next step was clear. RNA interference had to be made to work in a test tube reaction so that it could be dissected piece by piece. Tehre were bound to be enzymes in the cell that helped the strands target specific gene messages for destruction. Those needed to be discovered as quickly as possible to keep pushing the research forward. Along with another MIT professor, David Bartel, Dr. Sharp put two postdoctoral students on the job immediately.
The news was spreading fast. At the Cold Spring Harbor Laboratory, an independent research institute on Long Island, Gregory Hannon learned in a meeting that gene-silencing seemed to work in fly embryos as well. "I got on the phone with my lab and said, "This is a general phenomenon, get the fly cells out of the freezer right now," he remembers.
Though Dr. Hannon had been working on cancer genes, he now dropped everything in order to mash fly cells to make the liquid cell "extract" needed to start sifting through the new reactions' biochemical components.
It was also natural to wonder if the technique could be used in human cells. But there was a roadblock. The kind of molecules created by Dr. Fire - long, double stranded RNA molecules - were known to be toxic to animal cells. The big molecules triggered the cells' sophisticated defenses against viral invaders, throwing them into a panic mode and causing them to commit cellular suicide.
But then, British plant scientists found a new clue - tiny bits of double stranded RNA floating in the cells of Dr. Jorgensen's petunias as well as other plants. It looked as if the big strands were being diced into tiny ones.
The next move was obvious to everyone: Both the MIT group and Dr. Hannon raced to search for these small strands of RNA in their fly extracts.
Credit for such findings would go to whoever published first. Phil Zamore, one of the MIT students who had gone on to start his own lab at the University of Massachussetts, says he'd found the small RNA's already when he heard through the grapevine in late February 2000 that Dr. Hannon had similar results. "That was a huge race. I hope never to be that stressed out again," he says, recalling how he pulled together his manuscript in a week of 18 hour workdays.
They published withing days of each other the following month, but Dr. Zamore told the more complete story. The small RNAs were guiding the silencing reaction. If their sequences were programmed to match a gene, it would shut it off almost completely.
The finding made sense. Big double strands of RNA were being chopped into smaller ones, amplifying the effect. Dr. Hannon later identified the enzyme doing the chopping, which his lab dubbed 'Dicer." Like a multiple warhead, the smaller segments were each homing in on messages being sent by the target gene, then calling in enzymes to destroy it.
How Genes Make Protein
How to silence a gene
That finding suggested it was possible to overcome the immune-response obstacle. "The small RNAs are critical, because if you inject those, they no longer induce the reaction. People are not going to keel over because of a massive viral response," says Dr. Fire.
This summer Dr. Sharp's lab showed that gene-silencing seemed to slow the growth of HIV in laboratory cultures. And with researchers at Stanford University, Dr. Hannon studied gene-silencing in mice whose livers had been engineered with a gene from a firefly - lending a whitish glow to their organs when viewed through a special microscope. After designing small RNAs to target the gene, called luciferase, they reported that the mouse livers lost as much as 98% of their glow.
Last week, Dr. Sharp and his collaborators raised $15 million to form Alnylam, Inc., which aims to develop treatments against hepatitis and cancer. Dr. Sharp in 1978 helped fund of the world's first biotechnology companies, Biogen, Inc.
But the commercial landscape is already becoming crowded, and with scores of patent applications being made, people in the industry predict that scientists will eventually have to haul out their lab notebooks to prove exactly what they knew and when.
Although teams such as Dr. Hannon's and the MIT researchers have been first to publish many of the most important results, there has also been much research going on behind the scenes. "They did a great job on the science, but with respect to the patent application, they are later than us," says Stephan Limmer, a Bavarian biochemist who has raised $4 million from the German government and investors to back Ribopharma AG, the company he and a colleague formed in 2000 to start working on drug treatments.
MIT says it thinks its own patents will stand up and is looking to resolve the situation amicably.
Meanwhile, the discovery of the molecular trigger for gene-silencing is starting to unleash major new insights into what the human genome is actually doing and tying together a number of loose threads in biology.
The biggest find now emerging is that the human genome appears to carry code for hundreds of small RNA molecules of its own. Dr. Zamore reported in Science last week that these molecules could use the cell's silencing machinery to shut off other genes. In doing so, they are probably controlling aspects of embryonic development by, for instance, repressing the activity of genes responsible for the formation of the brain or limbs once their jobs are completed.
The process appears to have still other roles. In plants, it;s known to protect against viruses, which hijack cells in order to carry out massive, unapproved copying of their own genes. Dr. Fire thinks gene-silencing is part of an ancient game of genetic hide-and-seek between cells and viruses, which carry their genes in the form of RNA. "Watching a toddler get virus after virus, you'd think this was working all the time," says Dr. Fire.
Many of these effect now seem to be at work in many forms of life, including plants, animals and fungi. "The fact that these have been conserved in evolution means they have very important roles, says MIT's Dr. Bartel. "It looks like small RNAs have been shaping gene expression since the beginning of multicellular life."
Incidentally, I don't have much problem with evolution of plants, either scripturally or scientifically. That may just be because I have not studied it as much as I have animal or cellular evolution. I do know that they are prone to polyploidy and other things that could in theory make macro-evolutionary changes easier.
How do you figure that? It seems to suggest the flexibility and adaptability of the genome, creating more possiblities, rather than problems, for evolution.
It certainly shows an additional means by which the organism corrects for mistakes, intrusions, and defects. It is interesting that this seems to be a low level - and very specific - attack on genetic errors. Both viruses and mutations create genetic changes and this system seems to be able to dispose of both of them. This is a more specific system than the immune system which basically directs the attacks throughout the body.
It should be noted also how the scientists in this research call this a 'program' several times. Kinda shows some intelligent design in knowing exactly where the problem is and solving it in an economical way.
I guess it depends on how you look at it. With gene silencing you have a dramatic change by subtraction -- an order is given via RNA to stop a protein production and create a new characteristic.
Information must be added to a genome -- wings, thumbs, sexual reproduction --for common descent to be true. Dramatic changes in species is considered to be evidence of common descent.
That dramatic change is caused -- in some cases anyway -- by subtraction is an argument against the common wisdom.
But understand that in no way I'm claiming this disproves your position.
I found this research totally fascinating also. It is very important in many ways. It shows the existence of double-stranded RNA, something not seen before. It also shows a new, very specific, gene suppressing agent. This is good for the organism, which can fix problems in an efficient manner. However, it also gives a tremendous help in genetic research. It enables us to 'disable' a gene and see what happens. It is much more efficient than other methods which is why it has been adopted so quickly by research firms. We can expect to see many new discoveries as a result of this technique in the near future.
You'd be a great hero to many of us here!
Yeah, that particular kind of "intelligent design" is generally referred to as "darwinian evolution". Surely even you don't have any difficulty with short pieces of RNA, much less than a hundred bases long, evolving randomly? As you note yourself the immune system requires more extensive mixing, matching, mutation and selection than that to produce its specific antigens.
That's what FR is for. Only we identify and dice up leftist doubletalk, instead of double-stranded RNA.
Well, that is the evolutionist explanation, but of course there is another explanation. That the function was intelligently designed and that is why it is found apparently just about everywhere in both plants and animals. Evolutionists often claim that the reason something old is still around is that it was 'evolutionarily preserved'. There is no reason to say such a thing except evolutionary bias. An intelligent designer would not constantly reinvent the wheel, but would use what already worked elsewhere in new designs.
What is interesting here is that this adds another method by which the organism tries to prevent changes to it in addition to others we already knew about. This makes it harder for the mutations needed for evolution to be true to succeed.
Completely untrue. Gene silencing by the RNAi pathway would have no effect on the vast majority of mutations. The pathway is effective against exogenous double-stranded RNA, such as is produced in the life cycles of some viruses, and perhaps against the self-complementary inverted repeat sequences found in the transcripts of retroviruses and retroposons. These are features which specifically differentiate RNAi targets from endogenous RNA transcripts. The underlying principle is similar to innate (non-adaptive) immunity, which leverages distinctive molecular signatures of invaders such as bacteria and parasitic wroms. Indeed, conceptually it may be appropriate to think of RNAi as a particular type of innate immunity.
Well, there are indications in the article that point very much to intelligent design:
The small RNAs were guiding the silencing reaction. If their sequences were programmed to match a gene, it would shut it off almost completely.
You did notice the word 'programmed' there did you not? Have you ever heard of a program, regardless of the length that was written at random? Note also that they have to match the gene which is to be silenced. Note also that " the extra copy of the pigment gene he'd added was somehow cueing the plants to shut off their purple color" so we have the gene checking and knowing that something was amiss and issuing double-stranded RNA to the specific gene to stop its protein production. Note also that double stranded RNA is not normal, so we have a special function to do this which needs a special code to do it.
It is even more complicated than that however. We must remember that the genome is just a long series of DNA. This DNA only has four possible meanings and when read in threes as in genes it only has some 64 possible meanings. So how does this DNA know in the case of genes it is to produce single-stranded RNA and in the case of these silencers to make double stranded RNA? The answer is pretty obvious: that the DNA in the genome is primarily a program of which the genes are really the exception, they are the data used by the program. The rest are instructions telling the organism what to do. It is the only possible explanation for the genome 'knowing' that something is amiss, where it is amiss, what RNA is to be produced and where it needs to be produced.
And yes I do deny that any kind of long string of information even a few hundred bases long can arise at random just as I deny that a bunch of monkeys can write even a short sonnett or a short piece of programming code.
Completely untrue. Gene silencing by the RNAi pathway would have no effect on the vast majority of mutations. The pathway is effective against exogenous double-stranded RNA, such as is produced in the life cycles of some viruses, and perhaps against the self-complementary inverted repeat sequences found in the transcripts of retroviruses and retroposons.
You say that it is not true that it prevents some mutations and yet you give examples of some mutations which it prevents! So regardless of the semantics of the matter it is part of the array of methods which an organism has to prevent mutations. It should be noted also that both viruses and cancers often work by inducing hyper-replication of cells and this directly fights this problem.