Slide 1: This slide shows plasmid PSC101 carrying the frog gene in a bacterium. (Plasmid is a piece of circular DNA; DNA is deoxyribonucleic acid, which is the building block for genetic material.) This is an electronmicrograph, which magnifies thousands of times. This circular structure is a bacterial plasmid. Inserted into this bacterial plasmid is a frog gene. Incidentally, P stands for plasmid and SC stands for Stanley Cohen and 101 is the number of plasmids produced in this lab.

Construction of recombinant DNA

Slide 2: The next slide shows, in more graphic detail, how this plasmid, from the bacterium, was altered to

incorporate a gene from a frog. At the left is plasmid Stanley Cohen #101. At the right-hand top is the gene from Xenopus, which will be inserted into the bacterial plasmid. (Xenopus is the scientific name for a frog.) A certain enzyme is used to cleave the bacterial plasmid and open up the ring, as illustrated here. The same enzyme is utilized to dissect out the gene from Xenopus, which is then inserted into the bacterial plasmid. This process closes the circular confirmation of the bacterial plasmid, and the result is what is now referred to as recombinant DNA. In other words, the Xenopus DNA, or the Xenopus gene, is now combined with bacterial DNA from E-coli. Escherichia cold is the gram-negative bacterium present in the lower GI tract of all mammals. This recombinant bacteria carrying the frog gene proceeded to express, along with its normal repertoire of bacterial genes, a frog gene as well. (Recombinant is when DNA from one source is recombined with DNA from another source; gene expression refers to production of the protein encoded by the gene.) Never before had such a genetically altered organism existed.

Slide 3: This is an electronmicrograph of the first genetically engineered organism produced by Bayer and Cohen in 1973. As mentioned earlier, these bacteria contain a frog gene.

On the basis of this pioneering work, a new science has developed called genetic engineering. Genetic engineering constitutes the ability to manipulate DNA to the extent that it can be cut, rearranged, or amplified, and then inserted into another organism. Recombinant DNA technology is possible because of the universality of the genetic code among organisms as diverse as bacteria, elephants, and pine trees. Thus, organisms classified in different kingdoms have the same gene chemistry code. (Genetic code refers to the sequence of nucleotides; nucleotides are chemical molecules containing deoxyribonucleic acid; these are the building blocks of genes.)

I would now like to return to the subject of biotechnological advances in medicine. In 1982, the U.S. Food and Drug Administration approved the use of the first genetically engineered medicine, namely, recombinant human insulin. Recombinant human insulin is made by bacteria. When we think of bacteria, we think of disease, illness, bad smells, waste, and tonsillitis. However, biotechnology has harnessed the metabolic activity of bacteria to produce medicines for humans. In the past, diabetes was treated with pork insulin. (Diabetes is a disease where there is an abnormality of sugar metabolism.) The insulin was purified from the pancreas of pigs. One disadvantage of pork insulin was that some humans were allergic to pork insulin, because it is not chemically identical to human insulin. However, the insulin that is made by the bacteria is made with the human gene and, therefore, the insulin made by the bacteria is chemically identical to the insulin made by the human pancreas. Furthermore, it is much cheaper to make human insulin by genetic engineering than it is to extract porlc insulin and purify it from pork pancreas.

Slide 4: This slide shows how large amounts of insulin can be obtained through genetic engineering.

On the left of this slide you see human chromosome #2. (Chromosomes are organelles in the nucleus that contain the genes.) The gene for insulin is cut out of chromosome #2 by specific restriction enzymes and inserted into a bacterial plasmid, as illustrated. The bacteria containing this plasmid then produce insulin, which can be separated from other bacterial proteins to treat diabetes. The bottom left shows the insulin gene being inserted into the bacterial plasmid. The plasmid is then inserted into the bacteria, in this case Escherichia coli. A culture of these bacterial rods is shown in the middle of the slide. The broth from these bacteria contains insulin. The insulin is purified by special techniques and is then used to treat patients with diabetes. E-coli cultures crank out millions of molecules of insulin. This is a much more efficient process than purifying insulin from pork pancreas.

Other products produced by genetic engineering and used in human medicine include proteins to dissolve blood clots; growth factors that stimulate white blood cell production; erythropoietin, which stimulates the production of red blood cells; human growth hormone for the treatment of short stature; interferons and interleukins, which are used in the treatment of viral diseases and some cancers; monoclonal antibodies, which are used to deliver cancer drugs to specific cancerous cells, and many other factors.

Thus far, we've talked about making medicines by genetic engineering, but what about altering the genome, as in the case of Mrs. Calabash's unborn daughter? In 1990, the first attempt was made to use genetic engineering to combat genetic disorders.

Slide 5: This slide shows two children with a genetic disorder of the immune system successfully treated with gene therapy. These two girls suffered from an immune disorder requiring each of them to live in a microbe-free bubble because of a defective gene of the immune system. Doctors extracted bone marrow cells from the girls and replaced the defective gene with a normal gene. These genes were then returned to the girls' bone marrow, where they began to produce a normal immune protein. Both girls are now well and healthy and living normal lives outside, the sanitized bubble.

Sometimes, the trickiest part about gene therapy is getting the plasmid DNA into the host cell. Recently, an instrument called the gene gun was developed to actually shoot the gene into the host cells.

Slide 6: This slide shows how the gene gun could be used for gene therapy for bone diseases.

Thus far, we have mentioned that biotechnology has made advances in medicine. Another area where advances have been made is forensic work. The first time police used DNA fingerprinting on a murder suspect, the test cleared an innocent man of the charges. (DNA fingerprinting is not really a fingerprint but, rather, a metaphor for fingerprinting; DNA fingerprinting is a molecular technique to show differences in DNA structure, which are unique to a given individual.) In 1986, police in Narborough, England were convinced that a suspect had committed two murders. The suspect had even confessed to one of the murders. However, DNA fingerprinting proved that he had not committed either murder. Police had collected and tested blood samples from every possible suspect, which included more than 4,500 men in

Sometimes, the trickiest part about gene therapy is getting the plasmid DNA into the host cell. Recently, an instrument called the gene gun was developed to actually shoot the gene into the host cells Narborough and a nearby town. The DNA fingerprinting identified the real murderer, who was convicted of both murders.

Slide 7: This slide shows DNA from several individuals cut with an enzyme to generate a mixture of DNA fragments which are then separated by gel electrophoresis. (Gel electrophoresis is the technique that is used to separate the fragments of DNA based on their size; different individuals show different patterns.) (The bands consisting of the largest DNA fragments move at the slowest rate and, thus, are found at the top of the gel.) Each column represents a different individual. Because the genetic make up of each individual is different, you see here different DNA fragment patterns among different individuals. By analyzing the size and the intensity of the bands, one can establish the identity of the individual.

It is extremely unlikely that any two unrelated people would have exactly the same DNA fragment pattern on gel electrophoresis. Thus, DNA fingerprinting can provide indisputable evidence of identity. As of now, there are no uniform government-supervised standards for DNA testing and, thus, DNA tests are often challenged in court as unreliable.

DNA fingerprinting techniques can be applied to different biological specimens, including blood, semen, bone, and hair.

Slide 8: How can genetic engineering be used to the advantage of agriculture?

This slide shows Dr. Paul Umbeck and his genetically engineered longer, stronger, cotton fibrils.

Another big breakthrough in agriculture has been to develop crop plants that are resistant to the herbicide glyphosate, a powerful biodegradable weed killer. Glyphosate kills most actively growing plants by destroying an enzyme needed by the plants.

The trick was to have the herbicide glyphosate kill off the weeds but not the crop plants. To do this, genetic engineers found bacteria that made this enzyme even in the presence of glyphosate. Genetic engineers then introduced this glyphosate-resistant gene into crop plants. This gene was introduced into soy beans by a special genetic engineering method.

Slide 9 This slide shows soy beans genetically engineered to resist herbicide treatment. On the left, you can see soy bean plants that have been treated by glyphosate. They show no ill effects from this herbicide treatment. The dead or dying remains of soy bean plants not genetically engineered to resist this herbicide are shown on the right. The genetically engineered soy bean crops now do not need to be weeded. Instead, the farmer simply treats the field with glyphosate. All growing plants die except the crop, which is resistant to glyphosate. Glyphosate is quickly broken down in the environment and, thus, is a big improvement over most herbicides.

Another big advantage of this whole process relates to one of the biggest problems in agriculture -- the loss of fertile topsoil to erosion. The current program of herbicide resistant crops should markedly decrease this erosion, since, without weeds, it is unnecessary to intensively cultivate croplands, and, as a result, there will be less erosion.

In addition to many advances in agricultural crops, genetic engineering has resulted in agricultural advances in livestock production. For example, the size of beef cattle has been increased as a result of genetic engineering. In addition, agriculture and medicine have combined forces to produce what is called a transgenic animal. Such transgenic animals have been used to make medicines.

Slide 10: This slide shows a transgenic sow that produces protein C in her milk. By a special genetic

engineering technique, the Protein C is only made by milk-producing cells. Human protein C is an anti-thrombotic protein that is used in coronary bypass surgery to reduce bleeding and, thus, the amount of donor blood needed. It is much cheaper to produce therapeutics in livestock by this transgenic means than by bacteria.

Slide 11: In addition to making transgenic animals, one can apply genetic engineering techniques to clone animals. In fact, the cloning of Dolly, a sheep in Scotland, made headlines

throughout the entire world. (Clone means genetically identical.) This slide shows a photograph of young Dolly with her surrogate mother.

Dolly doesn't look like her mother, because she is genetically a clone from another sheep and is genetically unrelated to her birth mother.

Slide 12: This slide illustrates the strategy that made Dolly. The first step is to perform in vitro fertilization. The Scottish doctors,lead by Ian Wilmut, first removed cells from the udder of one ewe. Next, they obtained an

unfertilized egg from another ewe and removed t e nucleus from this unfertilized egg. The third step was to fuse the udder cell, which contained DNA, with the unfertilized egg, which did not contain DNA, because the nucleus had been removed. This fusion, in effect, produced a fertilized ovum, which contained only genetic from the ewe that provided the War cell. This ovum was then placed in the uterus of a third ewe, the surrogate mother. The surrogate mother later gave birth to Dolly, which represents a clone of the ewe that supplied the udder cell.

This sounds like a simple procedure, but it is far more complicated than I have depicted. In actual fact, in order to make Dolly, 277 cloning attempts were required. The advantage to cloning an animal, from an agricultural standpoint, is that, in each herd of animals, there are always a few animals that stand out as being superior to others, either in milk production, as in the case of dairy cattle, or in beef production, as in the case

of beef cattle. With cloning, one can take the best animal of the herd and make several clones of this animal and thereby improve the overall quality of the herd.

Slide 13: This slide illustrates the types of experiments being done in a genetic engineering laboratory. "How disappointing-they don't appear to have grown at all."

In conclusion, genetic engineering provides the means to produce enormous advances in the fields of medicine and agriculture that can improve the health and nutrition of the world’s population, provided Mrs. Calabash and her neighbor agree.