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MANIPULATING GENES


Gene transfer or genetic engineering involves the transfer of genes from one species of organism to another species, i.e.from a donor into a recipient organism. It is seen by some as a simple extension of other biotechnological processes, whereas to others it is considered as a development with much more sinister implications. In fact there has been pressure to use the term biotechnology, which has gained some public acceptance, to cover both.

A gene is a unit of hereditary information (i.e. it normally passes on characteristics from one generation to another), and is composed of DNA.

Gene manipulation may be advantageous because it makes the resulting genetically modified ortransgenic organism easier to grow or manage, or to transfer a characteristic to a different crop, etc.

It differs from selective breeding which only involves members of the same species, in that usually only single genes are moved, often in addition to that organism's normal complement of genes ("genome"). Because selective breeding involves the normal methods of sexual reproduction (gamete transfer, fertilisation and development, etc.), it only results in large combinations of genes being transferred (the haploid number of chromosomes contained in a gamete is in effect half a genome), and the effect of these genes may be masked or diluted due to dominance by other genes.

Gene transfer techniques

In the laboratory, specific enzymes may be used to cut andsplice DNA:

Restriction enzymes break DNA at specific parts of the molecule (nucleotide base sequences) - usually leaving so called "sticky ends". This can be done to both DNA from which genes are being taken, and to DNA in which genes are being inserted.

DNA ligase enzymes may be used to rejoin such sections into the other DNA.

The DNA containing the selected gene for the desired characteristic may then be inserted into cells of the target organism by means of vectors (here used in the service of Man, not disease organisms).

There are 2 main types of vectors:
plasmids and viruses (see previous notes on micro-organisms).

Gene transfer using plasmids


Edexcel Agrobacterium tumefaciens is a bacterium which in nature causes plant disease - "crown gall disease" - but only in some dicots ("broad-leaved plants"), not in monocots (grasses and cereals).

A gall is a mass of undifferentiated plant tissue - similar to a cancerous tumour - produced in response to such an infection. Crown galls are usually produced on the stem just above the surface of the ground.
The bacterium contains a section of DNA called a plasmid in addition to its usual component of DNA. This Ti (tumour inducing) plasmid normally incorporates its DNA into the cells of the plant host ("integrating with their genome").

The ability of this organism can be utilised in genetic engineering to insert other genes into crop plants.


Sources of genetic material


It is thought that there is no technical reason why any characteristic in one species which is thought to be potentially useful in another species cannot be transferred by the application of these principles.

However, it is said that, due to commercial pressures, the main use of gene transfer to date has been to confer resistance to pests or diseases, rather than more direct impact on yield or other desirable characteristics. A gene thought to be useful may be obtained from a variety of sources, e.g.:-

The gene for resistance to herbicide (weedkiller) may be obtained from (occasional) weeds which survive treatment with this chemical. This could perhaps usefully be incorporated into a crop which would then benefit from reduced competition from weeds, less hoeing etc, when sprayed with the appropriate herbicide. Commercially it would also mean that the seed and herbicide would be part of the same supply deal.
This procedure has actually been applied to crops of commercial significance, e.g. soya (beans), sugar beet, tobacco, and oilseed rape.

The Bt gene for production of insecticidal toxin fromBacillus thuringensis has been incorporated into several crops in order to protect them against insect pests.

Protection of crops from insect damage has also been tested using the gene for venom from scorpions!

Similarly, the effects of incorporating pest resistance genes from snowdrops into potatoes has been investigated.

Other novel ideas include the transfer of genes coding for important animal proteins such as the hormone insulin into plants, such as potatoes, which are easily grown and processed, and the transfer of genes into easily managed animals such as cows, sheep, etc, which may produce milk containing valuable proteins such as human antibodies and anti-cancer products.

Edexcel Other alternative approaches involve isolation and modification of genes so that normal developmental changes do not occur. For example, there are several enzyme-controlled stages in the ripening and subsequent deterioration (spoilage) of fruit. Modification (inhibition) of the genes producing these enzymes can slow down the changes which occur after fruit is ripe. As a result, the keeping quality or shelf-life of the fruit is increased, and possibly the quality of products derived from these fruits is improved, as well as reducing the processing costs.
This has been achieved and licensed in the case of tomatoes and products such as puree.

It is also said that attempts are being made to produce strains of soya beans which will flourish in temperate climates, and which are tall enough to facilitate mechanical harvesting.


Commercial implications of genetic modification of organisms

Interestingly, several biotechnology companies working in the field have attracted the attention of investors excited by the prospect of profits to be made. However, much venture capital has been used in the process, and there is considerable commercial rivalry and secrecy as to the exact details of the processes. Similarly, there is much public distrust as to the true intentions of workers in the field, and campaigners on each side have raised the profile of these activities in relation to regulatory authorities.

Recently there have been a variety of developments:

- Organisations representing consumer interests wish to ban GMOs (genetically modified organisms) from entering the food supply chain, or to have it kept separate from other food supplies, and have its origin specifically stated in the product labelling

- Supermarket chains have in some cases responded either by sourcing supplies of non genetically modified foods, or by identifying such ingredients in the labelling of the food, even if only a minor constituent. This is an ongoing development! Iceland was one of the first to do this, and on the 28th April 1999 Tesco also announced it was stopping using GMOs.

- Growers or importers have mixed genetically modified foods with non genetically modified foods, either on the grounds that to do otherwise would increase costs, or in order to confuse the issue, in the hope of speedy acceptance of the product. This has been the case with soya beans, which are a major export from the USA.

- Test plots of varieties of plants being assessed for future use are covered by a variety of regulations designed to reduce the likelihood of any transfer of genes to surrounding crops. In some cases corners have been cut and tempers have run high. Some pressure groups have advocated a moratorium on these trials, i.e. postponing them for several years.

- Farmers and growers must sign undertakings not to save seed from the crop for use to start another crop next year, because agrochemical companies have patent and other rights to the varieties used, and expect an exclusive agreement to use a combination of seed and control chemicals from the same supplier.
- The impartiality of some of the more important committees overseeing trials carried out by large companies has been called into question. Many of the decisions used to be made by employees of companies with interests in genetic modification, and a company owned by Lord Sainsbury, a government minister, holds patent rights to, and therefore profits from, important techniques in genetic manipulation.



Stages in the process of genetic modification - in more detail

There are several methods of introducing the gene into the target organism.

Plasmid
Gene removal
DNA from the donor organism is broken up into short lengths with "sticky ends" using a restriction enzyme.
One or some of these fragments should include the gene for the desirable characteristic, but often there is an element of chance, so the procedure is frequently repeated many times.

The tumour inducing plasmid which consists of DNA from Agrobacterium tumefaciens is similarly treated with the same restriction enzyme, opening out the circle of DNA leaving 2 sticky ends. The presumed gene DNA is then mixed with the plasmid DNA, and conditions provided for the DNA ligase enzyme to work. In a number of cases, this will result in the plasmid re-joining, but with the gene incorporated into it.

Fullof
The plasmid is reintroduced into the bacterium, which can then be grown up in large numbers by standard microbiological methods. When plants are infected with these bacteria, they will form galls of undifferentiated tissue, some cells of which will contain the required gene.

Sections of the gall may be encouraged to grow by special plant tissue culture techniques, possibly bulked up in the lab before conditions in the medium are changed to encourage growth of roots and shoots.

The resulting small plants may eventually be potted up and finally transferred to the field!

Other potential applications

The Agrobacterium situation has several parallels with symbiotic nitrogen fixation.

This also involves the activities of a species of bacterium (Rhizobium leguminosarum) which enters a plant organ (root of a legume) resulting in a change in the plant cell growth to form a root nodule, in which bacteria grow and perform chemical transformations.

It is hoped that genes for nitrogen fixation (nif cluster) may be transferred to non-leguminous plants. However there is more genetic information in these (12/20-30genes than can be easily transferred using plasmids, so more ambitious methods are being tried. Gene expression (turning them on) is a problem, especially as bacteria (prokaryotes - lacking nuclei/chromosomes) differ greatly from higher plants (eukaryotes - chromosomes protect DNA inside nuclei).

Viruses as vectors


Edexcel Certain viruses can infect cells of animals and plants "without completing a destructive cycle" so they may also be used as gene vectors. They can usually carry larger portions of DNA than plasmids can .

An example is Lambda (lambda) phage - a bacteriophage which can modify bacteria. DNA from a so-called temperate phage becomes incorporated into the DNA of its host: the bacterium Escherichia coli (E. coli), and can remain there indefinitely without having any harmful effect.

The phage DNA can be opened using restriction enzymes and foreign DNA may be inserted, so that the viral DNA can integrate with the host cells's "chromosome" (it is then called a prophage), and replicates with it at cell division.

Similarly, plant viruses may be used to transform plant cells genetically.

Other methods of gene transfer

These more drastic methods are mostly used with plant material, because the cell wall forms a barrier.

Ballistic techniques
Minute tungsten particles are coated with the DNA to be inserted, then shot into the target cells with an explosive charge. Apparently, this does not, however, cause significant structural damage to the cells.

Electroporation
In this technique, a brief pulse of electric current is passed through the cell, temporarily increasing surface permeability so that DNA is taken up from the surrounding liquid. This has been especially useful with pollen tubes and has resulted in the genetic transformation of seeds. Certain chemicals may have the same effect on the permeability of the cell wall.

Genetically modified organisms and food production
Edexcel

The same techniques used in the production of insulin and antibiotics may be applied to the use of genetically engineered bacteria in food production. Examples include yeasts with high alcohol tolerance, microbes with enhanced ability to digest waste straw, peat, coal, oil, etc., and improvements in capacity to produce valuable substances e.g. enzymes, flavourings, colourings. To some extent, industry has favoured the application of genetic modification processes to organisms which have achieved public acceptance, such as yeasts and lactic acid bacteria (Lactobacilli), which are responsible for cheese production as well as yoghurt and soy sauce.

Transgenic animals

Edexcel

There are obvious advantages in transferring genes for characteristics which are seen as desirable in the agricultural context, such as resistanceto common animal diseases, lack of horns in cattle, and more efficient growth conversion, e.g. due to higher production of growth hormone, or greater digestive efficiency.

However, potential human medical applications have been seen to offer great opportunities. Production of blood clotting factor (needed by sufferers of the genetic condition haemophilia) can be induced in the milk of sheep. So-called "designer milk" containig low cholesterol could probably find a profitable market.

More controversially, it has been said that transgenic organisms such as pigs could be used as sources of organs for transplants into humans, if human genes were transferred into these organisms at the embryo stage. This could reduce problems of rejection due to the immune system of the donor. However, the risk of transfer of potentially very serious virus diseases from one species to another has become more obvious in the light of scrapie/BSE/CJD which is said to have "jumped the species barrier".

These possibilities pose many ethical dilemmas.



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