The Lowly Fruit Fly Takes a Giant Swat!
10.29.08 by Syam Anand
by Syam Anand
Who would imagine that scientists are mindlessly wasting taxpayer dollars on stupid fruit flies? That too, not the money that came through the NIH after peer review, but from ear marks- those wasteful “pork-barrel” projects that help politicians channel money for projects in their pet constituencies! Is it not a shame that this has been going on behind our backs until the most popular governor in USA decided to bring out this activity into the open in her first major policy speech?
During her first major policy speech, she made fun of the lowly fruit fly and the researchers who are after it. Amazingly, she even managed to garner a few laughs from the audience.
Watch the portion of the speech for yourself here.
http://www.youtube.com/watch?v=HCXqKEs68Xk
Poor fruit flies! Also, those poor souls of scientists who worked and continue to work on them!
Outrageously ignorant- that is how I would summarize the fruit fly remarks.
This is what happens if you don’t believe in science. This is exactly what happens if you don’t understand how science works. This is what happens if you don’t know that “WHAT IS TRUE FOR E. COLI IS ALSO TRUE FOR THE ELEPHANT”. This is what happens if you don’t understand evolution (whether you believe in it or not and to what scale). The list is long. But since it starts sounding personal, I will get back to the point.
The point is lowly organisms such as fruit flies have contributed more than what one can wish for when compared to studying “real patients”. Who could imagine that another low-life, in fact an even better description would be a “life-less thing” such as the T4 bacteriophage- a virus that infects E. coli- would lay the foundations of modern molecular biology and genetics? The answer is- those scientists who thought ahead of their time and understood the potential of systematically understanding how small “things” work in order to understand how big things also work. This is based on something called the universality of rules (with some exceptions for the sake of argument). Even laymen will get it, provided they keep their eyes and ears open. This is as simple as the laws of motion being the same in physics irrespective of what model you are using for your studies.
The fact is that studying fruit flies is exactly the way to accelerate research to understand how brain functions. I don’t know if fruit flies have a soul! But I sure know they have brains, even though they are tiny! In fruit flies, you can knock-out (functionally or physically remove) individual genes and proteins and ask questions about how it affects brain or any other bodily functions. And the fly would not complain, right? You can’t imagine doing that with so much convenience and economy in humans (even the dumbest person would agree). In fact, fruit fly research has identified components that affect not just brain function, but also developmental and genetics defects, thus helping scientists to extend these observations to other model systems and human beings.
FYI GOVERNOR: “About 75% of known human disease genes have a recognizable match in the genetic code of fruit flies (Reiter et al (2001) Genome Research: 11(6):1114-25), and 50% of fly protein sequences have mammalian analogues. An online database called Homophila is available to search for human disease gene homologues in flies and vice versa. Drosophila is being used as a genetic model for several human diseases including the neurodegenerative disorders Parkinson’s, Huntington’s, spinocerebellar ataxia and Alzheimer’s disease. The fly is also being used to study mechanisms underlying aging and oxidative stress, immunity,diabetes, and cancer, as well as drug abuse.”- CITED FROM WIKIPEDIA!
At this rate, the governor would argue to stop supporting research using mouse or guinea pig models, or the coveted Danio rerio (zebra fish), which is again another low-life not worthy of studying. Check out the following video, which is an example to understand how studying these low-lives help improve human health.
http://www.youtube.com/watch?v=ZItgyfuxsfM
There are similar countless examples in scientific literature.
If it is really true that a Senator or Congressman really used an earmark for funding fruit fly research, I congratulate that person for showing the courage to invest money for the future of public good. Flexibility is good, especially for funding science as there are too many ideas out there, which you cant clearly bet on, unless you give it a chance. That is what makes this country strong and a leader in fundamental and applied science. Hope better sense prevails in people. I hope laymen and CIPs (curious and interested people) educate themselves adequately from reliable sources before falling prey to misinformation campaigns about certain scientific investigations.
Long live T4 phages, fruit flies, zebra fishes, darwin’s finches and all low-lives!
Syam Anand
Pittsburgh, USA
Does Open Source Apply to Science?
10.24.08 by Syam Anand
Release early, release often- the mantra of open source softwares- can it be applied to Fundscience?
By Syam Anand
I want to first introduce the mantra of open source softwares to the small minority of those who may not be aware of it. “Release early and release often” summarizes the philosophy of people who work and support open source softwares. Open source softwares thrive on the committed efforts of groups of people who work from different parts of the world. They put their brains together for a common goal- constant improvement based on feedback. They are not part of any real organization. But they always evolve into an organization of sorts that is governed by operating principles that co-evolve with them keeping in tune with changing priorities. The good thing about evolution is, it tests and selects the fittest. Being more flexible thus makes them more adaptable as an organization.
From a strategic perspective open source softwares thrive on “real” feedback and “real” solutions. People who actually uses these softwares work on it and to improve it and keeps on improving it. First, they don’t wait to come up with packages such as version x or version y and then try to sell it in a form that cannot be tinkered with (similar to a biology kit whose information in “proprietary” and you don’t have access to it, even if you own the kit!). Second, they don’t set the intervals with which they come up with updates, beforehand. They do it on a regular basis. If a software has a bug, it is explained in the open for possible solutions. When a solution is found, it is notified to everyone. As a practicing scientist, it seems very similar to what we do everyday for every aspect of laboratory life, except for funding. We regularly do experiments, we regularly improvise and find better ways to ask questions and get answers. But when it comes to funding, we can do it three times a year for NIH and once a year for the foundations. Of course, we regularly work towards getting funded!
When you talk to any scientist (established or beginner) about funding avenues that are currently available, one constant complain you hear is that it takes a huge amount of time to get a proposal reviewed and funded. This is true for NIH (since I am a biologist, I would restrict myself to NIH) and foundations that fund research. By huge I mean, upto ONE WHOLE YEAR! Herculean efforts, planning and lots of luck seem to be a requirement to survive the transition periods. Consider the rate at which funding is granted nowadays- 10% of grants that require substantial amounts of preliminary evidence (which in turn takes money, time and manpower to generate!). This means if one has to rewrite and resubmit, which takes another year, a few lives and careers will be on the line and soon off the line. And it actually happens in real life. A glaring example is that of Dr. Prasher who did not win the nobel for GFP because he ran out of funds and had to give his GFP clone to two other scientists who went on to win this year’s nobel in chemistry. It seems he is driving a courtesy shuttle now! (read the blog by dgaddy in fundscience.org on GFP). This brings me to the point I wish to make- fund early and fund often is the way to go, if anyone wants to be different and make a difference in the way research is funded.
Fund early and fund often is easier said than done, as one has to consider the availability of funds, availability of reviewers and the time constraints this would place on the reviewers. Since the success of open source strategies depends on cooperation between individuals who are knowledgeable, building an interactive community and providing a forum for concurrent evaluation of proposals are the starting steps. The next step would be to remain flexible. Too many rigid rules for submission, evaluation and granting funds would make it look the same. Make it simple and flexible and let it evolve.
If there is a possibility of funding one project every month, I would rate that as more rewarding for supporting science ON TIME, rather than 12 grants at the end/beginning of every year. Fund early and fund often. If fundscience can adapt this mantra, it will address one low point of every funding agency that I know- TURNAROUND TIME. This is a niche that fundscience can evolve into and make its own habitat. How to achieve this objective is a matter of discussion, debate and planning. But this is one requirement that is yet to be a major focus for a scientific funding agency. One argument against this would be that competition is not going to be uniform in every month. My answer to that is that competition wont be uniform no matter how it is done, as scientists come up with fresh ideas regularly and there is no clear way to mark a genius from a dumbo other than wait and see how and what he/she does in a relatively long period of time. There is no point in holding a science Olympics to see who wins!
As a closing argument, I feel that if a scientific paper can be reviewed in two weeks time (accelerated papers take this long) and a suitability report for a journal (such as science/ nature) can be obtained overnight from their editorial boards, a short write-up (one-three pages) can be read and reviewed and put to vote within a month. Another fresh beginning would be to put the submitted proposals for a vote not just by the “experts” (for which there are other forums such as NIH and the innumerable foundations) but by everyone including Ph.D students, post-doctoral fellows, doctors, engineers and people who are curious and interested to participate. If you can vote for electing your president, you could vote on the science that you wish you fund directly too! The role of experts will be to moderate the discussion and voting in a fruitful and non-partisan manner.
As for which projects can be funded (since the public is directly involved, this is a concern for a lot of people), the institutional review board and research administration to which the scientist making the proposal is answerable will ensure that all rules and regulations are met with and research is conducted ethically and responsibly.
Syam Anand
Pittsburgh, USA.
Bug’s Life
10.20.08 by Syam Anand
Bacteria are ubiquitous microorganisms (typically in the scale of a few micro meters; a micrometer is one ten thousandth of a centimeter) that are present in almost every habitable nook and corner of earth. Some of them have even specialized for surviving seemingly uninhabitable places such as hot water springs and extremely dry or acidic environments. In an era of high personal hygiene what would amaze most of us is that there is an abounding presence of these minute life-forms in our own bodies. It is estimated that in our own bodies they outnumber our cells nearly one to ten! Most of these harmless bacteria live on our skin and inside our digestive tract. Among the advantages of harboring these bacteria are nutritional and immune system-stimulatory functions. This suggests that they are useful to us in many ways than we previously thought plausible. However, under certain circumstances such as immune depletion, these harmless bacteria can become dangerous and even fatal to the host. One notorious example is Staphylococcus aureus, which lives on our skin. It is estimated by CDC (Centers for Disease Control and Prevention), USA that S. aureus causes more infections than AIDS! Worse, this bug seems to be able to develop resistance to any drug that is thrown at it from time to time. Thus we have Methicillin Resistant Staphylococcus aureus (MRSA) and Vancomycin Resistant Staphylococcus aureus (VRSA) among others that make a substantially long list.
Watch the following video to get a grasp on the “grapes of wrath”.
More information about MRSA and the challenges it pose, is available in the following video.
So the question is what does one do if a superbug keeps developing resistance to every known antibiotic? The answers are not simple. In fact, it requires concerted action at all levels of our community as it involves both personal and policy initiatives. There are some simple steps that the public can do such as washing hands and taking care not to share personal stuff through which these bacteria spread most of the time. As for doctors, a simple step such as washing their hands after attending individual patients would decrease the risks of spreading the infection from one patient to another. Hospital management could step in and try to keep high-risk patients in isolation as S. aureus has been reported to flourish and spread in hospital environments. As for scientists like me, we have to up our ante to discover more viable targets and increase the available arsenal against these bugs. We should also try to take our discoveries from the “bench-top to the bed-side” by actively collaborating with the drug industry. Those of us with business acumen could even don the entrepreneurial hat ourselves. The demands for an ever-growing arsenal is always high in order to succeed in this fight. This is also true for a lot of other bugs as well, such as multi drug-resistant tuberculosis and AIDS.
In the beginning, screening for antibiotics was a relatively simple but laborious process where people hunted for fungal samples from soil for anything that kills bacteria in culture. In nature, several fungi produce antibiotics as means of efficiently competing with their smaller cousins for survival space, in this case soil. In the period that followed, people have successfully modified naturally occurring compounds isolated from such screens-such as penicillins-to increase their efficacy as more and more drug-resistant bugs evolved. However, we seem to have run out of steam with these approaches.
Fortunately, scientific advance provided us with alternatives. By screening synthetic combinatorial chemical libraries (such collections often contain several thousand compounds) and structure-based design principles, we can design drugs that specifically target essential proteins present inside these bugs. However, it is not easy to predict targets and perform large screens in the absence of supporting basic research. Basic research into fundamental life processes in bugs is capable of providing additional valuable targets that can be exploited for therapeutic purposes. Unique metabolic pathways and essential proteins discovered by basic researchers should provide viable antimicrobial targets for future.
The recent discovery of a potent agent against MRSA is a glaring example of the triumph of basic research, interdisciplinary approach and the entrepreneurial attitude of one scientist who lead the effort. The research group led by current director of the Institute of Cell and Molecular Biosciences, New Castle University (UK), Prof. Jeff Errington, discovered the Achille’s heel of the superbug while they were studying it’s cell division machinery. During his studies, he noticed that the rounded shape of S. aureus made it highly susceptible to the inhibition of cell division unlike some of its bacterial cousins who had a more elongated shape. He then went on to exploit these findings by starting a spin-out company Prolysis Ltd. Recently scientists from Prolysis published their findings of a novel lead compound directed against the cell division machinery of S. aureus in Science (Science. 2008, 321, pages 1673-1675). Interestingly the compound has “potent and selective anti-staphylococcal activity”. A new company Nugenis Ltd is expected to take over the drug screening opportunities emerging from the Errington lab as Prolysis evolves into a drug development company. The case serves as one more classic example where the entrepreneurial spirit of a basic researcher is set to pay big dividends for public health by taking his discovery from the “bench-top to the bed-side”.
Syam Anand
Pittsburgh, USA.
PCR without the PCR machine!
10.9.08 by Syam Anand
Sounds like fiction? Guess not. It is a distinct possibility now. This innovation is likely to revolutionize field applications of PCR and further expand its commercial potential.
For those of you, who are unfamiliar with PCR: PCR stands for Polymerase Chain Reaction. Since 1983, when the idea of PCR was first conceived by Kary Mullis, it has grown to be the method of choice for economically amplifying fragments of DNA or RNA over a billion times with the help of polymerases- enzymes that make more copies of nucleic acids such as DNA and RNA. Due to its ability to make more of the same from very less of the starting material, PCR is the backbone of both basic-science labs and application-oriented labs such as biotech and forensics. The expiration of the original PCR patent is bound to bring in more innovations and cheaper methods from competing players in the multi-million dollar PCR industry!
Watch the following movie, which explains the molecular basis of the reaction in layman’s terms. In essence the two strands of DNA act as molds for making more DNA molecules that contain the same coded sequence information.
http://www.youtube.com/watch?v=_YgXcJ4n-kQ
Until recently, the PCR machine was an indispensable part of PCR reaction as it drives the temperature cycles in the PCR. Recently, scientists have successfully attempted to replace the PCR machine with helicases to achieve amplification of DNA. Currently PCR uses three different temperatures that cycle multiple times to accomplish amplification. The first step of denaturation melts the DNA by increasing the temperature to 94°C. In nature however, molecular machines called helicases carry out DNA melting. Helicases melt double helical DNA by using chemical energy provided as ATP. They hydrolyze ATP and utilize the energy released for mechano-chemical cycles that help them to physically separate DNA strands in a stepwise manner. Inside living cells, helicase reactions support a variety of DNA and RNA transactions.
There were a couple of challenges that had to be overcome to ensure specific and successful amplification of DNA with the help of helicases. Initially the process used a mesophilic (optimal temperature if neither too hot or too cold; typically between 30-37°C) version of a DNA helicase called UvrD from Escherichia coli (EMBO Reports, 2004, 5: 795-800). High temperatures increase the specificity of the annealing step in PCR. Therefore, in the next step, specificity was increased by using a thermophilic (high-temperature loving; typically between 50-72°C) version of UvrD helicase from Thermoanaerobacter tengcongensis (Journal of Biological Chemistry, 2005, 280: 28952-58). However the challenge of amplifying long stretches of DNA remained. Technically termed processivity, which is nothing but how long an enzyme stays and does its work on a molecule of DNA before falling off, this was another hurdle to be overcome. The longer an enzyme stays on the DNA without falling off, the longer it is likely to keep doing its job on the molecule. In this case, a highly processive helicase could melt long stretches of DNA and therefore help in amplifying long templates of DNA. This would increase the practical value of the technique.
The processivity issue was recently addressed by fusing the helicase to a DNA polymerase (Gene, 2008, 420: 17-22). Whereas the helicase alone could amplify only short substrates, its fusion to DNA polymerase (DNA polymerase by itself is so processive that it can copy the entire genome of E. coli, which is more than 4 million base pairs without falling off) made it much more processive. The helicase-DNA polymerase fusion can efficiently amplify DNA fragments upto couple of thousand base pairs length, which brings it into the realm of practical use.
Further improvements in processivity and specificity should see the technique finding wider use in biology laboratories. The technique christened Helicase-dependent amplification of DNA (HDA) would find applications in diagnostics and environmental monitoring by driving down costs and increasing its accessibility for applications in the field. HDA should be a huge benefit for people who do not want to routinely do PCR, to those who are not experts and also for whom investing in a PCR machine is not worth it. Along with the ability to detect amplified DNA by non-electrophoretic methods, HDA should make the PCR process user-friendlier. It would also increase the application potential of PCR-based techniques where there is a shortage of skilled personnel, especially in poorer nations. In the words of HDA’s original proponents, “the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care” should be around the corner.
Direct public funding of science
10.1.08 by Syam Anand
Currently, NIH funds the majority of biology research in USA using taxpayer dollars. The success of the NIH system and the adaptation of the scientific community to NIH have made this system stable and dependable. A significant amount of additional support for biology research, especially health related research, also comes from foundations. Some of them such as Bill&Melinda Gates foundation, and Howard Hughes Medical Institute provide substantial financial support far exceeding and often more attractive than what even NIH offers. A lot of good science have been done and fundamentally important discoveries been made by scientists who have been recipients of grants from all these sources. However, the success of NIH funding philosophy has influenced every major biological research funding operation that goes on in USA. Its impact is so widespread that the alternative funding options seem to be a product of inbreeding and incapable of providing new cultures or avenues that would complement the goals of NIH. As a result, the differences between most of these foundations that have clear independent goals are only superficial and all of them operate essentially on the same principles as NIH.
The NIH funding philosophy
NIH fellowships and grants come in different flavors with one underlying foundation, which is the provision of an efficient and goal-oriented institutionalized system that takes into account national priorities. This system makes sure that the available budget is prioritized and distributed between various sub-disciplines. Policy-makers arrive at these decisions through a systematic process, which tunes itself to changing demands from time to time. The institutionalized system makes sure that adequate resources are constantly available for the carrying out the funding process with a high efficiency by maintaining and providing trained pools of peer-reviewers (scientific colleagues) and program managers. The system of peer-review ensures that support is provided ONLY to scientists (or emerging scientists) who are likely to be successful. This is largely decided by three factors- 1) their prior success and pedigree 2) current environment of support and 3) the likely success of their research proposals based on preliminary evidence and arguments they provide in support thereof. The independent foundations that fund biology have also adopted the same principles and have not thought seriously outside the hat. So far, no one has argued with the success of NIH philosophy.
Alternatives?
So what is wrong with the current philosophy? Not much. The current system has provided everyone with steady and dependable advancement in science. In theory, there should be nothing to complain except that at unsure economic times (such as the one we are going through at present), NIH budget falls really short of demands and expectations of the scientific community. But a different kind of question is the following- is it so perfect that nothing different can be done? Not exactly. This is where Fundscience comes in. When David (CEO, Fundscience), talked to me about Fundscience’s objectives and how he plans to meet those, I was thrilled by the revolutionary nature of the philosophy behind it. To draw a parallel, it is like a constitutional amendment for the scientific nation, giving every individual a right to participate directly in science. Fundscience facilitates anyone to fund the science of his or her choice, directly.
Fundscience takes a giant philosophical leap from NIH and its inbred cousins. It primary goal is to tune the system to the time we live in. It is intended to let any ‘curious interested person’ (CIP; a term I am coining for the convenience of this discussion) to participate and bring the discussion and practice of science more into the open. One could say that it is built on similar lines as the successful philosophies of wikipedia and youtube. It attempts to bridge the conspicuous absence of a direct connection between a CIP and the scientific enterprise, which he/she is funding anyway! It proposes to build this bridge by taking out the common denominator of a seemingly inaccessible organization from the picture and putting a CIP in direct charge. It is as if the lobbyists and special interest groups have been removed from Washington! It is a community concept that is likely to succeed by creating a new niche where it does not clash with existing system and nicely supplements it.
Message for CIPs
For the science loving CIP, who wishes to take part in directly funding of his/her choice, Fundscience provides this avenue directly. The CIPs and trained scientists could work together on something that both are interested in. It could be anything ranging from something as complex and challenging as the study of cytoplasmic organization or something that could appear silly on the surface. This new philosophy would let people choose the science they want to fund by directly choosing from a list of scientists and available projects for funding. CIPs take the place of peer-reviewers! For the scientist who is interested in getting involved- convince a CIP and he might fund you directly! If a CIP is very rich, he/she could fund the science he/she loves directly. CIPs who are not very rich could come together to rate the various projects that are described online for funding. Then the projects could be chosen based on a public rating system for fun, importance to health, understanding of basics or specifics for which currently no funding is available, educational tools, exploratory tools, analytical tools, commercial applications…a long list that is left to be filled by the CIPs in charge. The possibilities are endless. Currently, the other available option for a CIP is to donate and loose track of his/her donation. If his/her donation were sizable, an attractive reward would be a building or a hall or a scientist named after him/her depending on the size of the donation. No much fun, right? I will bet that direct interaction and participation of the scientists and the community they serve would be a refreshing new way to conduct scientific research for both groups. In addition to acting as an additional resource-generating platform for scientists, it would also provide immense opportunities for public science education.
For the CIPs out there, this is what I (as an aspiring scientist myself) feel about Fundscience- Fundscience aspires to be the youtube or wikipedia for science funding. In a sense Fundscience could be your scientific vote just as youtube is your media vote! Depending on individual circumstances it will complement or supplement the University- and NIH-based funding avenues for science. With the participation of CIPs, Fundscience would make science a community enterprise. So CIPs out there- hop on Fundscience and become involved in some serious fun!
Biological processes: purpose or outcome?
09.26.08 by Syam Anand
Given below are dictionary meanings for three words, which are used routinely by biologists.
Purpose: the reason for which something is done or created or for which something exists.
Function: an activity or purpose natural to or intended for a person or thing.
Outcome: the way a thing turns out or the consequence.
Selfish DNA….hmmm, sounds like this kind of DNA has no purpose or objective in life other than some goal of its own! While other pieces of DNA are altruistic, providing coded information for the cell that harbors them, these selfish bits of DNA contribute nothing and at the same time ensure that they are passed on to future generations.
Selfish: lacking consideration for others. Concerned chiefly with one’s own profit or pleasure.
The questions I wanted to pose is the following: Is it right to use these kinds of expressions to explain the existence of a piece of a chemical because we don’t understand its function yet? Why not call it DNA of unknown function? Just like, genes in sequenced genomes with no apparent function are called FUN genes (FUN standing for function unknown). Does giving it any other name such as passenger DNA help? Are transposons more selfish than “selfish DNA’’? How selfish can DNA get? What is the scale for measuring selfishness in chemicals? Is Sodium chloride (common salt) also selfish? As sea water it got into my alimentary canal leaving a bad taste in the mouth and an upset stomach for my sensitive friends…all this to ensure that they get a free ride from Florida beach to Oakland, Pittsburgh doing nothing for me and my gang, when all other Sodium chloride molecules we ingested from the Cuban restaurant was making us feel good and also keeping our salt balance in the hot summer.
“The dogma is that teleology is unscientific, and in some contexts it is. Statements that the sun is for lighting the world, or that the moon is for calculating the date of Easter, have no place in science. But teleological language is often used by biologists, and can hardly be avoided except by circumlocution. Some biologists may regard it merely as appropriate shorthand, but for many of us it is the best way to convey what we have to say” * (Science, 1998, 281: p927).
Hence, another related question: Is a bacterium a mere manifestation of the sum total of all the reactions that take place inside of it? Is it an outcome without a purpose? Is the purported purpose just a result of interaction with its environment? Are there two ways to explain life of a bacterium? For example: a) bacteria live with a purpose such as multiply indefinitely, develop resistance to man-made antibiotics, infect and kill hosts whenever they get a chance OR b) is the life of a bacterium the outcome of all the chemical reactions that underlie its existence without any specific purpose. But the purpose we observe is just an outcome of its interaction with the environment?
I could make the question simpler by quoting “The main purpose of glycolysis is to provide pyruvate for the trichloroacetic acid (TCA) cycle, not to make adenosine 5-triphosphate. The glycolytic production of pyruvate reduces the cytosol by increasing the ratio of NADH [a reduced form of NAD+ (nicotinamide adenine dinucleotide)] to NAD+. Thus, glycolysis cannot continue without “something” returning the cytosolic redox potential to normal” * (Science, 1997, 277: 459-463). Really? So glycolysis has a purpose? Is it just one purpose that was put into glycolysis when it was assembled in its present form or did every glycolytic reaction have a purpose of its own and then all of them came together and decided on a larger common purpose?
How about something more simpler: The purpose of water is to sustain life on earth (everybody), is to dilute the coolant that is poured into the 21st century vehicle radiator (someone stuck with a can of concentrated coolant and a smoking car on a highway in the middle of nowhere), is to dilute the alcohol in your vodka (anyone?), is to solidify into ice at the artic poles (polar bears), is to fill large reservoirs so that golf courses can be made in a desert (Californians)…and so on and so forth.
Purpose in biological reactions, macromolecules and living systems? Is it not time we started talking about outcomes, especially when we are so close to making synthetic life in a lab from pure chemicals?
Finding answers to questions such as these is very important for expressing what we truly understand and report as science. Using the terms purpose (highly used), function (follows a close second) and outcome (very rarely used now) without sufficient thought puts us on a slippery slope.
<!–[if supportFields]> CONTACT _Con-3D61E5A91 c s l <![endif]–>Syam Anand<!–[if supportFields]><![endif]–>
Pittsburgh, USA.
*Disclaimer: These references are just two examples. There could be (and there are) several other references in scientific literature that supports the general argument I am trying to make here.
End of the “restriction enzyme era” in molecular biology?
09.15.08 by Syam Anand
Restriction enzymes are the workhorses of modern recombinant DNA laboratories. They cut DNA at specified positions by recognizing specific sequences on DNA (such as the order of G, A, T and C).
http://www.youtube.com/watch?v=-sI5vy-cD2g
Presently, the methods to engineer recombinant DNA molecules with predictable coding specificities rely mostly on the ability of these enzymes to cut DNA into predictable fragments, which can be subsequently ‘pasted’ in specific ways using DNA ligases thus generating new coding information. In the classic work that first described the generation of recombinant DNA molecules in E. coli1 and since then, the term recombinant DNA meant mostly the generation and use of mostly artificial combinations of DNA fragments through the use of restriction enzymes. In nature, the ‘real’ DNA recombination reaction drives evolution in incremental steps and it’s products are subjected to selection pressure due to its tie up with cell viability. Until recently and even as we write, molecular biologists the world over, are stuck with restriction enzymes for generating recombinant DNA molecules.
The current spectrum of our understanding of basic cellular processes such as how the genetic material is replicated and segregated into daughter cells, how damaged DNA is recognized and repaired by the cellular machinery devoted for this cause, how intracellular localization and trafficking of molecules take place and how populations of different kinds of cells (such as in the immune system or during the spread of cancer cells during metastasis) localize to various parts of the body by whole body imaging, to name just a few, have been driven to a large extent by our ability to interrogate these processes in creative ways with the help of mutational DNA libraries and gene fusions of various kinds. The economic generation of recombinant DNA molecules to answer complex biological questions is however limited by our ability to identify, characterize and commercialize restriction enzymes with novel specificities, even though the currently available enzymes has allowed us to do wonders. Manipulating DNA molecules that had no cut sites for user-friendly restriction enzymes, large genomic fragments where the enzyme specificities are represented multiple times or where the sites are not at desirable positions seemed to be nearly insurmountable barriers for path-breaking discoveries. It appears that DNA recombination can overcome these barriers in order to generate recombinant DNA molecules in a practical and user-friendly way.
One should mention here that recombination-driven generation of DNA clones would not come as a surprise for yeast geneticists. They have been continuously tapping the power of in vivo recombination to generate recombinant clones in vivo. Yeast cells generate recombinant DNA by Rad-51 mediated homologous recombination with high efficiency. All one needed was to simply transform yeast cells harboring the target plasmid with a dsDNA containing necessary length of homology at the ends and the desired sequence changes and wait for in vivo recombination to take over2. Biologists have also extensively used recombination-dependent cloning by exploiting the Cre-lox recombination reaction, which also did not involve the use of restriction enzymes or ligases3. However, the requirement of particular DNA sequences for the Cre-lox system (known as att sites) continued to act as a barrier for routine cloning experiments. Various groups have also utilized the lambda Red/ET recombination to create recombinant molecules in vivo. Thus, any DNA molecule with homologous ends (similar sequences) can be precisely joined together in certain strains of E. coli cells that express phage-derived protein pairs, either RecE/RecT from the Rac prophage, or Red?/Red? from ? phage4,5.
The recent discovery of a viral DNA polymerase that can pair DNA segments containing sequence homology (same DNA sequence) at their ends seems to be a revolution-in-the-making as far as the ability to generate recombinant DNA molecules in a tube goes. The underlying reactions that ‘paste’ the DNA fragments together do not employ classical recombination reactions that involve strand-exchange and DNA synthesis. However, it is more similar to recombination reactions than restriction enzyme/ligase-mediated cutting and pasting of DNA fragments. A single enzyme6 performs the reactions that search for homology at the ends of DNA and then join them together in a matter of few minutes. Fragments containing homologous sequences at the ends can be generated either by restriction enzyme digestion or PCR and pasted together in a matter of few minutes. Especially attractive is it usefulness for large-throughput screens where DNA fragments have to be shuttled between different DNA vectors. In future, it is possible that more efficient enzymes are discovered that carry out this reaction more efficiently. If the research community adapts to the new technology, this could be signal coming of the end of a grand era in recombinant DNA technology that was dominated by restriction enzymes.
One can envision large freezers stuffed with restriction enzymes of various specificities on the inside and buffer and compatibility charts on the outside, becoming a thing of past (just like the meter long sequencing gel apparatus, which appear now as if they are from the stone-age). Restriction enzymes however, would still have a place in biology and in our hearts and they will continue to do what nature intended them to do- protect the genomes of prokaryotes.
References:
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2. Wendland, J. PCR-based methods facilitate targeted gene manipulations and cloning procedures. Current Genetics, 2003, 44: 115-123.
3. Liu, Q., Li, M. Z., Leibham, D., Cortez, D. and Elledge, S. J. The uni-vector system, a method for rapid construction of recombinant DNA without restriction enzymes. Current Biology, 1998, 8: 1300-1309.
4. Muyrers, J. P. P, Zhang, Y and Stewart, A. F. Techniques: Recombinogenic engineering –new options for cloning and manipulating DNA. Trends in Biochemical Sciences, 2001, 5: 325-31.
5. Copeland, N.G, Jenkins, N.A. and Court, D. L. Recombineering: a powerful new tool for mouse functional genomics. Nature Reviews Genetics, 2001, 10: 769-779.
6. Hamilton, M. D., Nuara, A. A., Gammon, D. B., Buller, R. M., and Evans, D. H. Duplex strand joining reactions catalyzed by vaccinia DNA polymerase. Nucleic Acids Research, 2007, 35:143-151.
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