THE RECOMBINANT DNA SCENARIO.




a. The technique and the promise.
b. Asilomar Conference and Moratorium.
c. The New Guidelines.




THE TECHNIQUE AND THE PROMISE.



Paul Berg, Walter Gilbert, and Frederick Sanger were the discoverers of the recombinant DNA technique. They received the Nobel Prize in Chemistry in 1980. Their work depended on a number of earlier, unrelated discoveries.

(See The Recombinant DNA SCenarios: Andromeda Strain, Chimera, and Golem, by Bernard D. Davis, American Scientist, September-October, 1977.) Here is a listing.

1. The discovery of plasmids, the small, double stranded DNA rings in bacteria such as the Escherischia or E. coli. The plasmids contain opportunistic genetic information such as antibiotic resistance. When the cell dies, these plasmid rings become free and can be picked up by other E. coli cells acquiring the information.
2. Restriction enzymes can cut open the plasmid ring in a specific manner. The sequence of bases at the two open ends have a palindromic symmetry.
3. If foreign DNA is treated with the same restriction enzyme it will cut out DNA sequences, which will exactly fit into the open ends of the plasmid and under proper annealing conditions the ends will temporarily fuse by hydrogen bonding forming a closed ring once again.
4. The discovery of DNA ligases, another set of enzymes, different from the restriction enzymes, allowed the permanent fusion of the inserted DNA piece with the plasmid.
5. The uptake of the hybrid plasmids is very slow, but can be enhanced if the cells are heated in the presence of calcium chloride. Once inside the cell, the plasmids multiply, and can be recovered by selection techniques. From then on the products of the inserted DNA sequence or gene can be harvested.
The benefits of the recombinant DNA technique are immediately apparent. If the inserted DNA sequence is a human gene with a specific gene product, the material can be harvested efficiently in large quantities because the reproductive power of E. coli is very high and can be cultured in vets in large quantities. Under favorable conditions, E. coli divides every 20 minutes. This will result in large numbers in a very short time, close to 7 million per cell in 12 hours.

In conclusion, plasmids are very valuable tools in genetic engineering. The technique here described opens the way to gene therapy research, and to recombinant protein production, such as human insulin, growth hormone, and antihemophylic globulin.

ASILOMAR CONFERENCE AND MORATORIUM.



The outcry against the recombinant DNA technology was quite surprising. At first there was not much of a response, but soon, many people including some scientists, who were outstanding in their field, started to worry about the possible harmful consequences of the technique. There were two major area for concern. One was the idea that mixing of DNA segments from two totally unrelated organisms, such as mixing human genes with bacterial plasmids, creates an evolutionary monstrosity with unfathomable consequences. In the public domain, this idea was made profitable use of in the movie The Fly, where in a tele transporting device a human and accidentally a fly were transported and scrambled resulting in a tiny fly with a human head screaming “Help me! Help me!”, and a monstrosity of a human with a fly’s head and behavior. Of course, the whole thing was no more than an entertaining science fiction. The second was the idea of possible danger to human health. After all, when a multitude of restriction enzymes are used to chop up the human genome indiscriminately (the so called shotgun experiment), there is always the possibility of splicing a tumor producing human DNA sequence into a plasmid of an E. coli bacterium. If such recombinant E. coli is taken from the lab by a physical accident or by an accidentally infected lab worker, the incidence may end up by causing a terrible epidemic. Again the idea was made profitable by another science fiction movie The Andromeda Strain.

In the year 1975 a group of molecular geneticists, including the Nobel Prize winner George Wald, and other scientists, a moratorium was called for on recombinant DNA research at Asilomar. The conference was attended by the press. The Asilomar conference resulted in a set of guidelines severely restricting DNA recombinant work and rendering research extremely, and even prohibitively expensive. The guidelines restricted all recombinant DNA research to a single organism, the E. coli, and also demanded to carry out such work protected by four levels of physical containment, and by three levels of biological containment, which include the following. All recombinant DNA work is restricted to the K12 strain of E. coli, which has been isolated from a patient many years ago and since then transferred from culture to culture through endless generations. Since its origin the organism has established itself completely to a life in the artificial medium and it is doubtful that it could survive in its original environment. In addition, some mutations were also introduced, the EK2 strain, which renders it unable metabolize thymine, a constituent of its DNA, and diaminopimelate, a constituent of the cell wall. If these substances are not provided in the medium the E. coli cells are unable to multiply, and are unable to grow. These metabolites are not available in the human gastrointestinal environment. The E. coli used in recombinant DNA research is coded for self distruct outside the artificial medium of the laboratory.

The strange thing about all this is the fact that no harm has ever come from recombinant DNA research, while many accidental infections do occur in the everyday pathology labs in hospitals all over the country. And yet, the pathology labs never were targeted for such special, restrictive guidelines of biological containment. The recombinant DNA research somehow elicited unjustifiable fears in the public mind.
The fact is that E. coli cells living in the human digestive system ingest cast off human cells and incorporate some of the ingested human DNA into their own genome is an everyday occurrence. It has been going on for a very long time, and I do not believe that there are any possible combinations of bacterial plasmid and human DNA segments that have not already occurred many times over as a natural event. We humans and the E. coli have been evolving together for a very long time and the intimate association of E. coli with human cells has become well established. There are no monstrosities here but only natural events without harmful consequences.

As to the probability of incorporating a harmful human gene obtained through the shotgun experiment technique into some of the E. coli plasmids, and that the recombinant bacterial cell escapes from the lab and causes an epidemic, is zero. All we need to do is to figure out the probability of several simultaneously occurring events, each with a very low probability. Using the rule of multiplication, the probability of an Andromeda Strain vanishes into nothing.




THE NEW GUIDELINES.



The strict guidelines associated with the 1975 Asilomar Conference have been since relaxed considerably. The recombinant DNA research facilities at the Australian Deakin University may serve as an example. (http://www.deakin.edu,au/fac_st/bcs/Biosaf_Man/bsm_rdna.html)

“The Australian Government has determined that experiments involving organisms containing recombinant DNA from various sources be carried out under specified conditions. The government has established the Genetic Manipulation Advisory Committee (GMAC) to monitor such work. GMAC, and the University, are concerned not only with in vitro experiments with recombinant DNA, but with any operation which results in the construction and / or propagation of viroids, viruses, cells of organisms of novel genotypes produced by genetic manipulation, which are either unlikely to occur in nature or likely propose a threat to the public or the environment.”

The guidelines specify that each project of the kind listed in the above paragraph be registered with the University’s Biosafety Committee. The guidelines for small scale work with recombinant DNA require containment level PC2, which is in effect no more restrictive than ordinary caution in labs working with hazardous materials. The guidelines invoke Australia’s Standard AS2243 “Safety in Laboratories” Part 3 Microbiology. The purpose is to ensure basic levels of laboratory safety. There is no more reference here to extraordinary measures of the Asilomar era.

Note: Its seems that the whole story of public panic followed by the outcry, “Stop Research”, is being repeated again in the last few years. But now it is the genetically engineered agricultural products, which are under fire. The images of fear are very much the same: concern for health and concern for evolutionary and ecological consequences. It is very likely that this new panic will loose all foundation and will disappear just as the Asilomar guidelines did in their time.

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