Transgenesis and modifying gene expression
Transgenesis
- Transgenesis is transferring a gene from one species into another. The resulting organism is transgenic, or genetically modified.
- It differs fundamentally from selective breeding and cloning:
- Breeding and cloning work with alleles already present in the species.
- Transgenesis introduces a gene the species could not otherwise obtain, because the two organisms cannot interbreed.
- This is possible because the genetic code is essentially universal — the same base triplets specify the same amino acids in almost all organisms. A gene from one species can therefore be transcribed and translated correctly by another, which is itself strong evidence of common ancestry.
How a gene is transferred
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1. Obtain the gene. Two routes:
- Cut it from DNA using restriction enzymes, which cut at specific base sequences. Many leave sticky ends — short single-stranded overhangs.
- Make it from mRNA using reverse transcriptase, which synthesises complementary DNA (cDNA) from the mRNA. This is often preferable in eukaryotes, because mRNA has already had its introns removed, and a bacterial host cannot splice them out.
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2. Insert it into a vector. A plasmid — a small circular DNA molecule from bacteria — is commonly used.
- The plasmid is cut with the same restriction enzyme, producing complementary sticky ends.
- The gene and plasmid are mixed; complementary sticky ends pair by hydrogen bonding.
- DNA ligase forms phosphodiester bonds, sealing the sugar-phosphate backbone into a single recombinant plasmid.
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3. Transform the host. The recombinant plasmid is introduced into host cells — by making membranes temporarily permeable, by electroporation, or in plants by using Agrobacterium or a gene gun.
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4. Identify transformed cells. Only a small proportion take up the plasmid, so marker genes included in the plasmid allow successful cells to be identified and selected.
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5. Express the gene. A suitable promoter must be present so the host transcribes the gene, and in the right tissue if the host is multicellular.
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Applications include producing human insulin in bacteria, crops carrying a bacterial gene for insect resistance, and crops modified to synthesise a nutrient they otherwise lack.
Modifying the expression of existing genes
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The EN also covers investigation and modification of the expression of existing genes — changing when, where or how strongly a gene already present is switched on, without introducing a gene from another species.
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Why expression matters. Every cell in an organism contains the same genes, yet cells differ enormously. The difference is which genes are expressed. So altering expression can change an organism substantially without changing which genes it has.
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Methods:
- Altering promoters or other regulatory sequences, changing how readily transcription is initiated — so a gene may be expressed more strongly, in a different tissue, or at a different time.
- Gene silencing, in which small RNA molecules complementary to a target mRNA cause it to be broken down or block its translation, so the protein is not made even though the gene is present.
- Gene editing, which makes a targeted change to an existing sequence — correcting, disabling or altering a gene in place.
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An important distinction: transgenesis adds genetic material from another species; editing and expression modification change what is already there. The organism may end up with a genotype that could in principle have arisen by mutation or breeding, which is why the regulatory treatment of these techniques is debated. That debate belongs to AS91602 — here the point is simply that the two are biologically different operations.
Comparing the manipulations
| Selective breeding | Cloning | Transgenesis | Expression modification | |
|---|---|---|---|---|
| Source of genes | Within species | Single individual | Another species | Within organism |
| Speed | Many generations | Immediate | One generation | One generation |
| Precision | Low — whole genome reshuffled | Exact copy | Targeted, but insertion site may vary | Most targeted |
| New variation added | No | None | Yes | No new genes |
- The trend across the four is toward greater speed and precision. Selective breeding shifts an entire genome slowly and imprecisely; expression modification alters one sequence in one generation.
- That trend is the source of most of the biological implications, because the rate of change increasingly outpaces the rate at which populations and ecosystems can respond.
Biological implications
- Ecosystems. A transgene may move into wild relatives by cross-pollination, so a trait such as herbicide tolerance could spread beyond the crop. Traits affecting other organisms, such as insect resistance, may also affect non-target species feeding on or near the crop.
- Evolution of populations. A crop producing an insecticidal protein continuously applies strong, sustained selection to the pest population. Individuals carrying resistance alleles survive and reproduce, so resistance evolves — and the stronger and more uniform the selection, the faster it does.
- Genetic biodiversity. Widespread adoption of a small number of engineered varieties can displace local varieties and landraces, narrowing the genetic base available for future breeding.
- Health or survival of individuals. Modified organisms may show improved disease resistance or nutritional content; unintended effects on the organism's own physiology are also possible, since inserting a gene can disrupt sequences at the insertion site.
- Survival of populations. These techniques can also support survival — disease-resistant varieties can rescue a crop, and genomic information is used directly in conservation.
Worked Example
Worked Example
A crop is engineered to carry a bacterial gene coding for a protein toxic to a specific insect pest. It is planted across a large region.
- In the first five years, pest damage falls sharply and insecticide applications fall by about 80%.
- Numbers of a non-target insect that feeds on weeds within the crop also fall.
- By year eight, pest damage begins rising again. Sampling shows the proportion of pest individuals carrying a resistance allele has risen from under 0.1% to about 35%.
- Growers are advised to plant a proportion of their area with non-modified plants of the same crop.
Explain how the gene was transferred, and analyse the biological implications shown.
Answer:
How the gene was transferred.
The bacterial gene was isolated, using restriction enzymes to cut it from bacterial DNA at specific sequences, leaving sticky ends.
It was then inserted into a vector. The vector was cut with the same restriction enzyme, giving complementary sticky ends, which paired with those on the gene by hydrogen bonding. DNA ligase formed the phosphodiester bonds sealing the sugar-phosphate backbone into a recombinant molecule.
The recombinant DNA was introduced into plant cells, and marker genes allowed successfully transformed cells to be identified. Those cells were then grown into whole plants.
The plant can transcribe and translate a bacterial gene because the genetic code is essentially universal — the same base triplets specify the same amino acids in both organisms.
Analysing the implications.
1. Effect on the pest population — evolution in action.
This is the clearest result in the data, and it is a straightforward case of natural selection.
- Resistance alleles were present at under 0.1% before the crop was planted — so they existed already, arising by random mutation. The crop did not cause them.
- The engineered crop then applied an extremely strong selection pressure: pest individuals without a resistance allele died, while those carrying one survived and reproduced.
- Because the survivors were almost exclusively resistant individuals, their offspring inherited the alleles, and the frequency rose from 0.1% to 35% in eight years.
Why so fast. Three features combine: the selection pressure is lethal, so non-resistant individuals leave no offspring at all; it is applied continuously, because the plant produces the protein all season rather than being sprayed occasionally; and it is applied uniformly across a large region, so there is no refuge where non-resistant individuals survive to dilute the resistance alleles.
2. Effect on non-target species — an ecosystem implication.
Numbers of a non-target insect fell. Note that the data do not establish the cause, and saying so is important:
- It may be a direct effect, if that insect is susceptible to the protein.
- It may be indirect: with pest damage reduced and insecticide use down, weed management may have changed, reducing the weeds this insect feeds on.
Distinguishing these would require further investigation — feeding trials for a direct effect, weed surveys for an indirect one. Both are genuine ecosystem implications, but they call for different responses.
3. Why non-modified plants are recommended — analysing the strategy.
This is the most interesting part, and it follows directly from the selection argument.
A block of non-modified plants provides a refuge where the toxin is absent, so non-resistant pest individuals survive and reproduce there.
The biological effect is to maintain non-resistant alleles in the population. Resistant individuals emerging from the modified crop then interbreed with non-resistant individuals from the refuge, and if resistance is recessive, the resulting offspring are heterozygous and therefore susceptible.
This slows the increase in resistance allele frequency, because the refuge continually dilutes the resistance alleles being selected for in the modified crop.
Note what the strategy accepts. It does not prevent resistance evolving — it slows it, at the cost of accepting some pest damage in the refuge. It is deliberately sacrificing some yield to preserve the usefulness of the technology for longer, which is a trade-off rather than a solution.
4. What this shows overall.
The underlying implication is that these manipulations do not act on the modified organism alone. Introducing the crop changed the selection pressures on every organism interacting with it — creating intense selection on the pest, altering conditions for non-target insects, and changing the herbicide and insecticide regime.
The pest population evolved in response, exactly as evolutionary theory predicts, and did so within eight years because the selection was strong, continuous and uniform. The effectiveness of the technology and the speed of resistance evolution are the same property — a pressure strong enough to control a pest completely is strong enough to select powerfully for resistance to it.