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
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How to silence a gene
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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."
How? Why?
You have to remember Andrew, that evolutionists are great believers in miracles. Species transform themselves into new species how? It just happens! They mutate favorably - how? It just happens! They write long pieces of DNA specifically designed for special functions - how? It just happens! As Darwin said - if you have read through a few hundred pages of my blather already, you gotta believe the next whopper I am going to tell you.
I see, I am an idiot but you cannot point what I said that was incorrect even though you are by your admission a genius! Tell me another joke, I need a good laugh.
BTW, Gore, your firm opposition to the notion that species are capable of the slightest independent genetic change would suggest that you are a fixed species man. Most young earth, strict creationists allow for rather considerable genetic change, arguing, for instance, that the entire Equid family may well represent a single "created kind," even though all species of horses, asses, and zebras have different chrosome numbers, and therefore reorganizations in their DNA well beyond what you would countenance.
Creation science types generally take profound offense if they are stereotyped as holding to fixed species. Are you the embodiment of the elusive stereotype?
Fascinating! So, how much of the genome do you think is made up by these? And are there start & stop codons?
This brings up a question I have yet to get answered: What exactly are you measuring when you measure "information" WRT our bodies? The length of the genome, the number of genes, the number of proteins, or what? Here's an example of an increased number of coding sequences (the gene + later the disabling RNAi snippet) that cause a decrease in the number of proteins created.
Or looked at another way, you have a function (the gene) that previously would be activated when (A [the promoter region getting triggered] == TRUE), but is now activated when (A AND B == TRUE). That's a more complex expression that's being evaluated, but it would == TRUE less often than before. Is that a gain or loss of information?
Or looked at yet another way, if I start out with Snippet 1 below, and change it to Snippet 2, have I increased or decreased the information?:
// Snippet 1...
<script language="javascript">
oMsg = new String ("This is a message.");
if (oMsg != "")
{alert (oMsg);
}
// Snippet 2...
oMsg = new String ("This is a message.");
oMsg = "";
if (oMsg != "")
{alert (oMsg);
}
</script>
Unknown. This is a very new area of research, especially as it relates to mammals. The Scientist just published an article on a handful of scientists who now believe that much gene regulation is actually effected through untranslated RNAs transcribed from intragenic regions. This is, of course, a highly controversial idea. If so, short, interfering RNAs would be one of perhaps a number of RNA-based mechanisms for gene regulation. It's not controversial that RNAs do have certain very specific regulatory roles. For example, it's pretty clear that there are RNAs which play a role in X-linked dosage compensation in female mammals -- that is, where one of the two X chromosomes is effectively put to sleep in every cell to produce the same gene dosage as in males (XYs). However, it's generally thought that proteins are far more important than RNAs in regulating transcription in general -- but these scientists are suggesting there's a whole world of RNA-based regulation that we've only scraped the surface of to date.
In general, there is a general trend in molecular biology towards discovering more and more things that RNAs can do that we had previously only attributed to proteins. This is not that surprising: RNAs are just another type of polymer, and most biologists think that the first forms of life were based entirely on RNA, with RNA performing the catalytic role now largely taken over by peptides, and the genetic or information storage role now performed by DNA, in addition to serving its present primary role of messenger. However, no one knows for sure if the "RNA world" idea is correct, and there are some interesting alternative hypotheses out ther.
Getting back to RNAi, I don't think that is a legacy of RNA world, or anything quite as exciting as that. RNAi is based on a set of proteins which recognize double-stranded RNA -- something that isn't normally used in the cell -- and attacks it. So, it probably evolved as a defense mechanism against foreign nucleic acids. However, once the system was in place, it was co-opted to a gene regulation role, at least in a few instances that we have recently discovered, and possibly in a bunch of others that we don't yet know about. But that's something you see all the time in molecular biology -- a new module or function evolves, and then gets co-opted to other roles over geologic time.
My primary interest in RNAi is as a controllable method of knocking out specific genes, rather than in the phenomenon itself. We didn't know how to do this in mammalian cells until very recently, and it could turn out to be a powerful tool for reverse-engineering genetic circuits. Having said that, all the results aren't in yet. Check back in a a couple of years, and it may still turn out to be a lot of hype.
All those things could be used as metrics, but I don't think you could ever say one number is absolute and definitive, unless it's what you could compress a person's bitstream down to for a Star Trek transporter beam. Nucleotides of DNA in the genome, each corresponding to two binary bits, is a good rough measure, but there are a lot of caveats. For example, DNA never builds a cell or an organism by itself, so there is some information in the machinery of the cell, which isn't as easily quantifiable. OTOH, you could probably throw out much if not most of the DNA of a human being and it wouldn't make any significant difference to the development of the organism (provided you knew which bits to throw out!)
Here's an example of an increased number of coding sequences (the gene + later the disabling RNAi snippet) that cause a decrease in the number of proteins created.
Well, the use of an endogenous "siRNA" transcript to down-regulate another gene is conceptually no different from the use of a protein transcription factor to down-regulate another gene. It only really buys you something if the expression of the silencing agent -- the siRNA or transcription factor -- is conditional and regulated. If it were just cancelling out the effect of the other gene all the time, the whole thing would just represent a waste of bits, and would be lost over time, or never develop in the first place. And generally there also wouldn't be much point if the regulator has only one target, because then you just get a regress, since the target gene could just be regulated directly without the complication of the intervening regulator. But regulators are few and controlled genes are many: gene networks feature hierarchies of control, just like armies, nation-states and business corporations. One regulator controls many downstream genes, just as one foreman gives orders to several workers. These considerations hold regardless of whether we are talking about traditional protein-mediated control, or these novel siRNA-mediated mechanisms. Protein versus RNA is just a question of instrumentality, like JAVA versus C++.
I have never heard of any anti-evolutionist say that you can make hundreds of new DNA bases at random, so I think you are putting words in the mouths of others. I do not hold to fixed species in the sense you speak of either. Species have a very large gene pool. That's how we can get everything from wolves to chihuahuas to great danes from a single gene pool without any mutations.
I doubt very much that if this gene interference was a lot of hype:
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.
But that's something you see all the time in molecular biology -- a new module or function evolves,
That's a pretty big assumption being made by evolutionists considering no one has ever seen such a thing ever happening don't you think?
In the process of creating proteins, the DNA of course requires the use of raw materials from outside the nucleus located in the cell itself. These raw materials of course got there because when the cell was formed as a certain kind of cell its purpose was to get just those raw materials. That is why different cells have different functions and produce different proteins even though theoretically they could produce any protein at all since they all have the same DNA code.
What all this leads to however is a sort of chicken and egg problem - which came first the DNA or the cell? This is a really big problem for those who believe abiogenesis is possible.
One regulator controls many downstream genes, just as one foreman gives orders to several workers. These considerations hold regardless of whether we are talking about traditional protein-mediated control, or these novel siRNA-mediated mechanisms. Protein versus RNA is just a question of instrumentality, like JAVA versus C++.
Interesting that you mention programming languages in the discussion of gene regulation. You sound like an anti-evolutionist insisting that the genome is a program and was thus intelligently designed. Surely you do not wish to assert that programs are written and modified at random do you?
Every new discovery in the field of biology in the last century has shown us more and more complexity. It has also shown us more and more the interrelatedness of the different functions the organism. This is what intelligent design has always claimed is the case. This is why everyone before Darwin laughed off any suggestion that organisms could be built up piece by piece. Science is having a tremendous laugh at Darwin and the evolutionists fail to see that everyone is laughing at their silly theory.
All those things. Bascially, commands and data that didn't previously exist in the genome. In this case, information seems to exist but simply lay dormant.
Your code example (A [the promoter region getting triggered] == TRUE), but is now activated when (A AND B == TRUE) would indicate a programing change and an information increase. But suppose the code was written --I'm not a programmer so forgive the incorrect syntex -- as (A == TRUE) Unless RNA = Double THEN (A AND B == TRUE)?
That would not be an increase in information.
Hmmm... I was trying to reduce your desription to code, but I'm not sure exactly what you're trying to describe. (I can tell you're not a programmer. :-)Your code example (A [the promoter region getting triggered] == TRUE), but is now activated when (A AND B == TRUE) would indicate a programing change and an information increase. But suppose the code was written --I'm not a programmer so forgive the incorrect syntex -- as (A == TRUE) Unless RNA = Double THEN (A AND B == TRUE)?
That would not be an increase in information.
What I was describing was the logical expression that gets evaluated when the gene (G, we'll call it) gets activated & transcribed (condition A). If originally G got transcribed & eventually produced protein P, and then later a regulatory mechanism evolved that sometimes prevented P from getting produced, that mechanism could act at one of several points: It could prevent the activator from being produced in the first place, it could prevent the activator from reaching G & starting translation, it could prevent translation into P in midstream (like the RNAi seems to do), or it could block P somehow after it's produced. Either way it's a more complex system than the one without the regulation, even if the result is less P's getting produced in the cell.
glibertarians!
How did the various species of horsey's all get different chromosome numbers, considering that you obstinantly resist the notion of much more minor genetic changes than that even within species.
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