BioTechnology

BioTechnology

We have discussed some of the ways in which the structure of DNA can be changed in individuals through mutation and how DNA changes from generation to generation through recombination and independent assortment during meiosis and sexual reproduction.

For thousands of years humans have used selective breeding in agriculture, horticulture and what was once quaintly called "animal husbandry" to obtain and maintain desired inheritable traits with many species of plants and animals. In this sense, we have been manipulating genes for far longer than we have even known what "genes" are. We have taken advantage of the capabilities of many organisms to manufacture foods and beverages we like – yogurt making, beer and wine manufacturing and cheese are all examples of natural "biotechnology" which produces things we humans find useful. Drugs, such as penicillin are products of fungi, used for human benefit. The streptomycin drugs are bacterial derivatives. Penicillium mold was one of the first organisms deliberately "mutated" to produce better strains of the penicillin drug.

In addition, humans have, by our very treatment of our surroundings and those who inhabit our surroundings, been responsible (deliberately and/or unintentionally) for the loss of hundreds of species, and their unique combinations of genes by extinction...

Today, the field that we speak of as biotechnology, (the manipulations of organisms or their components to do something useful) or genetic engineering (direct manipulation of genes), effects changes in the DNA molecule and/or in the organism in very precise and directed ways, for research and for industrial or commercial applications.

Much of today's DNA research is based on refining natural methods of recombination, and taking advantage of some means of introducing new DNA into host cells. Such research is often called recombinant DNA research. We can now perform transgenic alterations, by splicing genes from one organism into a second.

Biotechnology or genetic engineering has many goals, including:

Improving our understanding of inheritance and genetics via basic research
Improving our understanding and treatment of genetic disorders (and some diseases)
Providing economic benefits in agriculture
Providing benefits (economic and health) in the production of biological molecules
Using the tools of DNA technology to increase knowledge in all areas of biology. For example, DNA homologies help us know more about the phylogenetic relationship of organisms
Facilitating criminal law via more accurate evidence analysis (e.g., DNA "fingerprints")

Some biotechnology research involves altering existing DNA to promote or prevent the expression of certain genes. One example of altered DNA is found in the commercial tomato industry. Biotechnology has been used to suppress the genes that promote ripening and softening of tomatoes so that tomatoes can stay on vines longer (to develop the proper flavor), but not get soft. Firm tomatoes are necessary for transportation purposes.

Many chemicals are now produced using strains of bacteria or fungi, genetically selected for their ability to produce quantities of the desired chemical, similar to the way the Penicillium mold was cultured to obtain a strain that produced good quantities of penicillin. It's much easier today, however, to find strains that produce the desired chemicals that it was in the 1940's.

The golden rice developed by a Swiss consortium is one example of using recombinant DNA techniques. This rice has genes to increase iron content, sulfur content, and B-carotene, as well as the enzyme phytase, which destroys the plant phytates that chelate iron. The goal of this project was to make a more nutrient rich rice, and targeted two nutrients, iron, and beta-carotene (a vitamin A precursor) that are lacking in diets of those who rely on rice as the diet staple in much of the world. Vitamin A deficiency is a leading cause of blindness in children in "third world nations", and is not reversible. It is estimated that 40 million children suffer vitamin A deficiency. Iron deficiency affects almost a quarter of the world's population. Distribution of this rice was withheld from the market for a number of political reasons (none of which in this case are monetary).

Much of the emphasis of this chapter in your text is to provide some information about the techniques that are used in Recombinant DNA research, and in Biotechnology in general. We shall look at some of those processes briefly.

A Closer Look at Recombination Techniques
Genetic recombination in eukaryotic organisms naturally occurs between homologous chromosomes with the following characteristics:

Crossing over occurs anywhere along the chromosome
Crossing over, except for mutations, is reciprocal
The crossing over distance is fairly extensive.

However, genetic recombination can also involve much smaller pieces of the chromosome that literally "jump" from one region of a chromosome to a second, or to a different chromosome completely. These so called jumping or mobile genes, or transposons, often work to inactivate genes in the region where they are inserted. Transposons were first discovered in corn by Barbara McClintock in the 1940's, where they inhibited pigment production of the kernels. She was awarded the Nobel prize several decades later for her discoveries.

Bacteria are particularly useful for research in biotechnology. Recombination in bacteria is common. Transformation, first discovered by Griffith, occurs naturally in many bacteria, and is a good example of recombination. Bacteria also have recombination using plasmids, small independent pieces of DNA incorporated into bacteria directly from the environment. A bacterium may have multiple copies of plasmids, and when the bacterium dies, its plasmids are released into the environment where they can be incorporated into a different bacterium.

Some bacteria have also have transposons, all of which carry a specific DNA sequence, called an insertion sequence that codes for a transposase enzyme. This enzyme allows for the insertion of the transposon's gene sequence into the bacterial chromosome. Transposons in plasmids facilitate the insertion of the plasmid into the bacterial chromosome. Since plasmids carry independent genes, new information is incorporated into the DNA molecule. DNA technology uses modified plasmids extensively to add desired genes to host cells for research purposes, and for the manufacture of chemicals needed by humans. Often plasmids that have antibiotic resistance, called R plasmids, are chosen.

Bacterial recombination can also take place by transduction, a process involving virus vectors, which can bring bits of DNA which were broken off from a previous host's DNA molecule when the virus left that host and add that DNA to a new bacterium. DNA can also be exchanged directly from one bacterial cell to a second, called conjugation.

Applications of Recombination Techniques in Genetic Engineering
Researchers in DNA Technology can take advantage of all of these recombination methods to get new DNA fragments into a bacterial host. One application of recombinant DNA today uses human "engineered" plasmids or virus vectors, and bacteria to create a "DNA library" of genetic material.

First the desired gene is located and isolated (which is why mapping the genome is so important). Once isolated, the target gene can be incorporated into a plasmid, and from the plasmid, added to a bacterium that can then synthesize the desired molecule or make multiple copies of a desired gene for basic research purposes, as well. The technique used for gene therapy, in which the gene of interest is spliced into a vector for distribution in human cells, is similar.

Recombinant DNA Technique

The process starts with using restriction enzymes to cut the DNA isolated from donor cells (not always an easy task) into fragments that can be identified, isolated and incorporated into a plasmid. There are many different restriction enzymes, each of which recognizes one specific nucleotide sequence.

Most restriction enzymes work by finding sections of DNA where the order of nucleotides at one end is the reverse of the sequence at the opposite end. This way a restriction enzyme can cut tiny sticky ends of DNA that match and can attach to sticky ends of any other DNA that has been cut with the same restriction enzyme. DNA ligase can join the matching sticky ends of the DNA pieces from different sources that have been cut by the same restriction enzyme.

Each time one cuts a set of DNA with a restriction enzyme and mixes the DNA with plasmids, thousands of different recombinant plasmids are formed. This conglomeration is called a DNA library. DNA libraries are important for genome mapping.

Isolating the Desired DNA from a DNA Library
Obtaining the desired gene to be incorporated into the plasmid is another task. A restriction enzyme cuts a number of different pieces of DNA, not just the one wanted. The trick is finding the nucleotide sequence for the gene we need from our library.

The variations in the DNA sequences between individual DNA samples that are determined by differences in restriction enzyme cleavage patterns are called restriction fragment length polymorphisms or RFLPs.)

These different DNA fragments can be separated using gel electrophoresis and then isolated for closer study. Often the RRLP may be within the target gene or at least near it. Gel electrophoresis is a common practice in gene analysis, for basic research, for medical research and for forensic study.

Gel electrophoresis separates charged molecules based on their molecular weight. An electric current is used to "drive" molecules that are placed in wells made in the gel from the negative electrode of the gel chamber toward the positive electrode. The rate at which molecules move through the gel is relative to their molecular weight. As the molecules are separated they appear as distinct bands on the gel. DNA fragments have a strong negative charge in neutral pH so they are well suited for the technique of gel electrophoresis.

Another way of locating a desired gene is using a DNA probe. A DNA probe is a small piece of single-stranded DNA or RNA with a known nucleotide sequence, and often a radioactive marker. A "probe" of radioactive synthetic single stranded DNA or mRNA can bind to its complement target on the DNA molecule pin-pointing the target DNA. This works only if a part of the target DNA code is known so that a "matching" probe can be used.

Once a gene has been located, researchers obtain multiple copies of the gene for their work. One method to obtain sufficient DNA is the Polymerase Chain Reaction (PCR). PCR is very valuable when trying to do a detailed analysis of a DNA molecule. PCR is also valuable when there is just a tiny amount of DNA from which to start. This is often the case when one is using DNA materials for potential evidence in criminal investigations, or when one is trying to reconstruct DNA from preserved and fossil materials. Kary Mullis won the Nobel prize in 1986 for his development of PCR. PCR uses alternating heating and cooling cycles starting with heated single-stranded DNA, primers that can join complementary DNA and DNA polymerase isolated from thermophilic bacteria to synthesize new molecules.

Applications of Biotechnology
The techniques of recombinant DNA technology can be used to compare genes from different individuals and different species or even map a species entire genome.

The human genome project, on-going for more than a decade, was deemed completed in summer, 2000. Genome projects include gene mapping using recombination frequencies and DNA restriction fragment mapping, restriction enzymes and DNA probes repeatedly along a chromosome with known markers, along with some special techniques.

Once a genome map is available, researchers can analyze the map. Genes are studied to identify nucleotide variations for gene disorders, allele differences, and to study the relationship of genes to gene expression. Genomics has been very useful for genetic diseases such as cystic fibrosis and sickle cell anemia.

We also use our knowledge of the genome to look for new gene sequences in different organisms. For example, the homeotic genes that affect developmental sequences in all animals can be isolated from a well-known species, such as the fruit fly, and then used as a DNA probe to see if a comparable gene is also in other animals, including humans.

DNA Fingerprinting
Another application is DNA Fingerprinting. A DNA fingerprint is the unique pattern that forms when an individual's DNA is subjected to a number of different restriction enzymes. Each recognizes and cuts RFLPs differently. The DNA is then run on a gel, and the patterns produced can be compared to target samples.

Because of the sensitivity of DNA analysis using these techniques, forensic cases can use DNA from small samples of skin, hair roots, semen, and even dried blood. If samples from the crime scene or from the victim match samples of the suspect, the suspect's presence at the crime can be shown. The accuracy of this technique for positive identification is very high. We can determine paternity in a similar fashion. A child's DNA fingerprint will be a composite of its two parents; DNA fragments that do not hybridize with its mother's DNA will match its father's DNA.

DNA fingerprints can also be used to match potential donors and recipients for medical purposes. DNA fingerprints can help establish evolutionary relationships.

Other Outcomes of DNA Technology
Agricultural Uses of Recombinant DNA Technology
Plant Uses of Biotechnology
Agrobacterium tumefaciens, a natural tumor-causing bacterium of plants, is the host bacterium for much DNA technology in plants. The tumor genes are removed, and a plasmid, the Ti plasmid, incorporates desired new genes. The altered Agrobacterium "infects" a tissue-cultured plant, which may express the inserted genes normally. There is much hope for this in agriculture.

Genes can also be "shot" directly into plant cells with a "gene gun". The gene gun injects coated DNA particles into the target plant cells.

Areas under study or in use today include:

Plants that can fix their own nitrogen, requiring less fertilizer (not successful yet).
Glyphosate herbicide resistance so crops can be sprayed with herbicides without damaging the crop plant.
Some of our crop plants have incorporated the protein from Bacillus thuringiensis (BT) produces a toxin in the intestines of Lepidopteran larvae. This has greatly minimized the need for some pesticides. BT has been engineered into the strains of bacteria (Pseudomonas) that invade root tissue, so that roots can also have protection against larval pests.
We mentioned at the beginning of this section the promise of golden rice, a transgenic rice that produces both iron and beta-carotene.
Tomatoes "engineered" to remain fresher longer.
Virus resistance has been "engineered" into some crop plants.
A common bacterium, Pseudomonas syringae, which lives on the epidermis of plant leaves and stems, causes ice crystals to form at temperatures above freezing. This bacterium has been genetically altered so it cannot make ice crystals; therefore plant surfaces do not freeze until much lower atmospheric temperatures are reached, an agricultural bonus.
Another altered bacterium helps Elm trees resist Dutch elm disease (which is caused by a fungus).
It is hoped by some to introduce vaccines into some fruits or vegetables, so that obtaining immunity will be easier than it is today. There has been success with potatoes, but they must be eaten raw.

A number of transgenic food crops have been approved. It is estimated that as much as 70% of the foods on our grocery shelves contain ingredients from crops that are genetically modified. Table 13-1, p. 252 lists them along with the traits that have been introduced.

Genetic Engineering in Animals
Growth Hormones
Bovine somatotropic hormone (BST, also known as BGH) has been successfully cloned and its use approved. This hormone increases milk production (something that is supposedly desired in spite of dairy subsidies that still pay farmers to produce milk in surplus) BST is also being investigated to see if it increases muscle development in cattle and pigs. We have a number of transgenic organisms into whose egg cells or early embryos a growth hormone has been injected. Such animals reach maturity much faster than normal so that they can be marketed sooner.

Selective Gene Breeding
Genetic engineering is widely being used for selective breeding in cattle, horses and other domesticated animals, saving many generations of breeding to get desired characteristics. Moreover, with cattle, at least, the young totipotent embryo can be "teased" apart so that one zygote can be used to make a dozen identical offspring.

With gene selection, one problem is that the insertion of the target has to be at a gene locus that can be "read" and must be inserted into gametes to be expressed in the whole organism, or at least into the target tissue area. In addition, the inserted gene cannot disrupt normal activity. Since there is little control over where the gene gets spliced into the host egg cells, such mutant or transgenic animals often have a low survival rate.

Biotechnology in Medicine
Genetic screening, as mentioned for sickle cell anemia and cystic fibrosis, is one technique that is used. Prenatal screening can help potential parents determine the risks of genetic disorders in their children, and perhaps, in the future (which is today) apply biotechnology to correct gene defects in the embryo.

In the1980’s researchers first succeeded in deactivating a good gene in mice so they could study the effects of a defective version. This has proved useful in studying specific genetic defects. This process is often called genetic knockouts. For example, such mice were used to study cystic fibrosis, Huntington's disease, Alzheimer's and some cancers.

Making Molecules
One outcome of DNA technology is that the desired DNA can be used to manufacture needed molecules. Although bacteria, and especially E coli, are used most, some products are made from incorporating the genes into yeast cells, or even mammal cells. Some successes are noted in Table 13.2, p. 256. Others include:

Gene Therapy
Stem Cell Research
Undifferentiated stem cells hold much promise in gene therapy to restore damaged and lost tissue. Embryonic stem cells (from the early embryo) are totipotent; no differentiation has occurred.

Mouse heart cells have been cultured from embryonic stem cells, and have been successfully transplanted into damaged heart tissue. There is promise that the technique could work in humans, too.

Stem cells are also found in differentiated tissues, and are called tissue-specific stem cells. As tissues differentiate in development, some cells remain, even in the adult, as stem cells. Bone marrow stem cells are used in leukemia therapy that involves bone marrow transplants. Success varies.

The gene needed for preventing severe combined immunodeficiency disorder has been successfully transferred to bone marrow cells of children who have this genetic disease. (The gene codes for a critical enzyme, ADA, needed to activate the immune system cells.) Stem cells from umbilical cord blood are harvested and the ADA gene is inserted using a retrovirus. Introduced into a genetically "impaired" child, the altered cells carry the needed gene to bone marrow, implant there and produce the needed enzyme.

Correcting Defective Genes in the Embryo
For the future we can expect similar techniques to be used in conjunction with gene corrections of "defective" genes. A couple with a known genetic disorder can provide fertilized eggs for culture. Embryos can be grown in culture along with a vector that carries the normal gene sequence. Genetically corrected nuclei can be extracted from the embryo culture and implanted in enucleated eggs from the mother. The genetically corrected egg can now be implanted and a "normal" child can result. It was recently announced that an embryo that carried gene markers for Alzheimer's had been "corrected" this way.

The Biochip
DNA technology can also be used to produce a DNA microarray assay that results in a gene microarray or biochip, a small but discrete collection of gene fragments on a chip. With DNA technology, these can be automated and give information about thousands of genes, all located on one biochip.

With the use of biochips, researchers can compare an individual's single nucleotide polymorphisms (SNPs) to the standard from the human genome project, or another biochip, identifying how the individual differs from the standard. Most people have a SNP about one per thousand nucleotides. SNPs may be responsible for many genetic differences, from cystic fibrosis and sickle cell anemia to red hair and cholesterol levels. A database of perhaps 300,000 SNPs is being developed and may be a future way for physicians to screen most accurately for genetic diseases. Your biochip may also uniquely identify all that is you, genetically speaking

Biochip analysis can also be used in cancer screening, for example, to better target a specific treatment to a specific cancer, not just for finding a genetic profile.

Concerns about DNA Technology
Some question the ethics of manipulating the genetic code which has evolved over billions of years just because we humans have reached a stage in our knowledge and technology when we can start to do so. Some of the questions we might ask are:

What happens if a gene splices into an inappropriate location in the target host's DNA or has negative effects on the host organism? What do we do then?

Will the genetic alteration affect only the target organism and with the target effect? Or might it affect some non-target organism deleteriously? For example, using genetic engineering in plants to provide pesticide and herbicide resistance means that we can grow crops with less use of the pesticides and herbicides that pollute our air, water and soil. However, pollen from the modified crops can spread to other areas, affecting the gene pool of the plant as well as impacting other organisms. Pollen from corn engineered with BT coats non-target plants in the area. Butterflies that feed on the non-target plants are then affected.

Should we have concern that, no matter how remote the likelihood, some altered organism might escape and cause grave harm to our ecosystems? After all, some natural organisms do just that.

In agriculture, there is hope of vastly improving plant productivity, by growing plants in areas not poorly suited for agriculture, such as saline soils, or soils with little moisture, or low fertility. What will this mean for the numbers of humans and non-humans who inhabit this earth now? What impact will this have on the earth's other resources? Not to mention the impact on that area not now suited for human agricultural practices.

With better screening ability, what are the ethical issues associated with screening for life threatening diseases? Should any genetic testing be accompanied by appropriate education and counseling so that families can make informed decisions about what they are discovering? There is always talk about insurance companies or potential employers using genetic screening to “screen out” those who might have expensive to treat problems.

As mentioned earlier, what about cloning and making more copies of certain individuals? Or choosing the genes one wants his/her children to have?

No matter how we address these types of concerns, the research conducted today is under very strict peer and national review, and oversight commissions. These rules were developed in the past, and as knowledge changes, rules and applications must also. Each of us might want to be as informed as possible so that we can contribute intelligently to the decision making processes.