Notes · BiologyUK · A-Levels

The control of gene expression

Every cell of an organism carries the same genes, yet cells differ because genes are switched on and off. This A-level chapter covers gene mutations and their consequences, stem cells and cell specialisation, the regulation of transcription and translation including epigenetics, the link to cancer, and the tools of recombinant DNA technology used to read, copy and manipulate genes.

6 sections~20 min reading time3 competenciesLevel Standard 1 · Advanced 5

T·0888 / 8

Exam profile

AO1 · Describe mutation, the regulation of gene expression and the tools of recombinant DNA technologyAO2 · Apply the effects of mutation and regulation to novel cases and interpret electrophoresis and expression dataAO3 · Analyse gene-expression and fingerprinting data and evaluate the ethics of stem cells, gene therapy and screening

Operators:describeexplaincalculateanalyseevaluateinterpretsuggest

basic level

This whole topic is A-level (A2) only; it is not part of the AS qualification.

higher level

The full A-Level expects the mechanisms of gene regulation, the link to cancer, and confident description and interpretation of the gene-technology toolkit.

Depth

Text

Contents · 6 sections

The control of gene expression

Gene mutations and their consequences#

AdvancedAQA 7402 3.8.1DfE GCE Biology - gene mutation

Types of gene mutation

Fig. 1Deletions and insertions cause a frame shift and are usually more damaging than substitutions.

A gene mutation is a change in the base sequence of DNA, arising spontaneously during DNA replication or induced by mutagens (such as ultraviolet light, some chemicals and ionising radiation). Because the base sequence codes for the amino acid sequence, a mutation can change the protein made, and hence the phenotype. Mutations are the ultimate source of all new alleles and therefore of genetic variation, so they are both the raw material of evolution and the cause of many genetic diseases and cancers.

The consequences depend on the type of mutation. A substitution replaces one base with another and affects only the one triplet in which it occurs; because the genetic code is degenerate, the new triplet may still code for the same amino acid (a silent mutation with no effect), or it may change one amino acid, or create a stop codon. A deletion or an insertion, by contrast, removes or adds a base and so shifts the reading frame from that point on (a frame shift): every triplet downstream is changed, usually producing a completely different and non-functional protein - which is why deletions and insertions are generally far more damaging than substitutions.

Whether a mutation has an effect on the phenotype also depends on where it falls. A mutation in a non-coding region (an intron, or DNA between genes) often has no effect, and even a coding mutation may be neutral if the amino acid changed does not affect the protein's function. This is why the degenerate code and the presence of non-coding DNA cushion organisms against many mutations, and it is a favourite examined subtlety - a mutation does not automatically change the protein.

Cancer is caused by mutations in the genes that control the cell cycle. Proto-oncogenes normally stimulate cell division; a mutation can turn one into an oncogene that is permanently active, driving uncontrolled division. Tumour suppressor genes normally slow division or trigger the death of abnormal cells; a mutation that inactivates one removes this brake. Either kind of change can lead to the uncontrolled mitosis that forms a tumour, linking this topic back to the cell cycle studied earlier.

Worked example

Comparing a substitution and a deletion

A coding sequence reads (in triplets) GCA-TTA-CCG. Compare the effect of substituting the first A for a G with the effect of deleting that same base.

Substitution
GCA -> GCG changes only the first triplet; because the code is degenerate this may still code for the same amino acid, so the effect may be small or none.
Deletion
Deleting the base shifts the reading frame: the sequence is now read as GCT-TAC-CG..., so every triplet from that point is different.
Conclusion
The deletion causes a frame shift and is likely to produce a completely different, non-functional protein, whereas the substitution affects at most one amino acid.

Result: The substitution affects one triplet (maybe none); the deletion causes a frame shift altering all subsequent triplets.

Typical mistakes

Saying every mutation changes the protein - a substitution can be silent, and non-coding mutations may have no effect.
Confusing the effect of a substitution (one triplet) with that of a deletion or insertion (a frame shift affecting all later triplets).

Active revision

Explain why the deletion of a single base near the start of a gene is likely to have a much greater effect on the protein than the substitution of a single base in the same region.

Active recall

Recall the key points — then reveal.

Sources: GCE AS and A level subject content for the sciences (Department for Education) · AQA A-level Biology 7402 specification (AQA)

Stem cells and cell specialisation#

StandardAQA 7402 3.8.2.1DfE GCE Biology - stem cells

Types of stem cell

Fig. 2Potency decreases from totipotent (any cell, plus placenta) to unipotent (one type).

Although every cell in an organism contains the same genes, cells become specialised because different genes are expressed in different cells - some are switched on and translated into protein, others are switched off. A liver cell and a nerve cell differ not in the genes they carry but in which genes they use. Stem cells are unspecialised cells that can keep dividing and can differentiate into other cell types, and they are classified by how many types they can become.

The four categories form a hierarchy of decreasing potency. Totipotent cells (such as the cells of a very early embryo) can differentiate into any cell type, including the cells of the placenta, and can form a whole organism. Pluripotent cells (embryonic stem cells) can become almost any cell type but not the placenta. Multipotent cells (such as the adult stem cells in bone marrow) can form a limited range of related cell types (for example, the different blood cells). Unipotent cells can form only one type - for example, the cardiomyocytes derived from a unipotent stem cell.

Stem cells have great medical potential because they can be used to replace cells and tissues lost through disease or injury - for example, treating type 1 diabetes (making insulin-secreting cells), spinal injuries, or blood disorders. Induced pluripotent stem cells (iPS cells) can be made by reprogramming an adult body cell to become pluripotent, which sidesteps some of the problems of using embryos and gives cells genetically matched to the patient.

The use of stem cells, especially embryonic stem cells, raises ethical issues that are examined as an evaluation. Using embryonic stem cells involves the destruction of embryos, which some consider unacceptable, while others weigh this against the potential to relieve suffering; there are also questions of safety (some stem cells could form tumours) and of consent. A good answer sets out arguments on both sides and reaches a reasoned, balanced judgement rather than simply asserting a view.

Worked example

Classifying a stem cell

A stem cell found in the bone marrow can form red blood cells, white blood cells and platelets, but no other cell types. State its category and justify your answer.

Identify the range
It can form several related cell types (the blood cells) but not any cell type.
Match to a category
A cell able to form a limited range of related types is multipotent (not pluripotent, which could form almost any type).
Justify
It is therefore multipotent - an adult stem cell restricted to the blood-cell lineage.

Result: It is a multipotent stem cell (limited to related blood-cell types).

Typical mistakes

Saying specialised cells have different genes - they have the same genes but express different ones.
Confusing totipotent (can also form the placenta and a whole organism) with pluripotent (cannot form the placenta).

Active revision

Explain why a bone-marrow stem cell is described as multipotent rather than pluripotent, and give one medical use of such cells.

Active recall

Recall the key points — then reveal.

Sources: GCE AS and A level subject content for the sciences (Department for Education) · AQA A-level Biology 7402 specification (AQA)

Regulating transcription and translation#

AdvancedAQA 7402 3.8.2.2DfE GCE Biology - control of gene expression

How oestrogen controls transcription

Fig. 3Oestrogen activates a receptor that acts as a transcription factor, switching target genes on.

Gene expression is controlled at several stages, most importantly at transcription. Transcription factors are proteins that bind to a specific region of DNA (near the start of a gene, the promoter) and either help RNA polymerase to bind and begin transcription (activators) or prevent it (repressors). Only when the right transcription factors are present is a gene transcribed, so the set of transcription factors in a cell largely determines which genes are switched on - and hence what kind of cell it is.

The action of the hormone oestrogen is the standard example of how a signal controls a transcription factor. Oestrogen is a lipid-soluble (steroid) hormone, so it diffuses through the cell-surface membrane and binds to a receptor that is itself a transcription factor. Binding changes the shape of the receptor's DNA-binding site, activating it; the oestrogen-receptor complex then moves into the nucleus, binds to the promoter of specific genes and stimulates their transcription. This shows how an external signal is translated into a change in gene expression.

Epigenetics is the control of gene expression by heritable changes that do not alter the DNA base sequence itself, but affect how tightly the DNA is packed and therefore how accessible it is to transcription. Two mechanisms are examined. Increased methylation of DNA (adding methyl groups to cytosine bases near a gene) prevents transcription factors binding and so switches the gene off. Decreased acetylation of the histone proteins around which DNA is wound makes the histones bind the DNA more tightly, condensing it so that transcription factors and RNA polymerase cannot reach it, again switching the gene off.

Expression can also be regulated after transcription, at translation, by RNA interference. Small interfering RNA (siRNA) is a short, double-stranded RNA molecule that is processed and then guides an enzyme complex to a complementary mRNA, which it cuts up (or blocks) so that it cannot be translated. This silences the corresponding gene at the level of the messenger, and it is both a natural control mechanism and a powerful research and potential therapeutic tool.

Epigenetic control

Fig. 4Increased methylation and decreased acetylation both switch a gene off by making DNA less accessible.

Worked example

Silencing a gene epigenetically

Explain how increased methylation of a gene's associated DNA prevents that gene from being expressed.

Methyl groups added
Methyl groups are added to cytosine bases in the DNA near the gene (often at the promoter).
Transcription factors blocked
The methyl groups prevent transcription factors and RNA polymerase from binding to the promoter.
Gene switched off
Without transcription factors, transcription cannot begin, so the gene is not transcribed and no protein is made - the gene is switched off.

Result: Methylation blocks transcription-factor binding, so the gene is not transcribed.

Typical mistakes

Saying epigenetic changes alter the DNA base sequence - they change how accessible the DNA is, not the sequence.
Getting methylation and acetylation the wrong way round (increased methylation and decreased acetylation both switch genes off).

Active revision

Explain how increased methylation of the DNA associated with a tumour suppressor gene could contribute to the development of cancer.

Active recall

Recall the key points — then reveal.

Sources: GCE AS and A level subject content for the sciences (Department for Education) · AQA A-level Biology 7402 specification (AQA)

Gene expression, cancer and interpreting data#

AdvancedAQA 7402 3.8.3DfE GCE Biology - gene expression and cancer

Expression of a gene in healthy and cancerous tissue

Fig. 5In this (illustrative) data the tumour suppressor gene is silenced and the oncogene over-expressed in cancer.

Cancer arises when the control of the cell cycle is lost, and both genetic mutations and epigenetic changes contribute. As seen earlier, an oncogene may become permanently active or a tumour suppressor gene may be inactivated. Epigenetic changes add another route: increased methylation of the DNA of a tumour suppressor gene can silence it without any change to the base sequence, removing the brake on cell division just as effectively as a mutation would.

A tumour may be benign (slow-growing and staying in one place) or malignant (invading surrounding tissue and spreading to other parts of the body by metastasis). Tumour cells differ from normal cells in dividing uncontrollably, in having an irregular structure, and often in producing their own growth factors and not responding to signals that should stop division - all traceable to abnormal gene expression.

Because some of the changes driving cancer are epigenetic, and epigenetic changes (unlike mutations) are in principle reversible, they are a promising target for treatment: drugs that reduce the abnormal methylation, or restore normal histone acetylation, may switch a silenced tumour suppressor gene back on. This is an active area of research and a good context for evaluation questions about new treatments.

A characteristic A-level demand is the interpretation of data on gene expression - for example, the amount of a particular mRNA or protein in healthy and cancerous tissue, or before and after a treatment. The skill is to describe the pattern accurately (using figures from the data), to relate it to the biology (for example, higher expression of an oncogene, or silencing of a tumour suppressor gene), and to be cautious in drawing conclusions (correlation is not proof of cause; sample size and controls matter). This is exactly the critical analysis that Paper 3 rewards.

Worked example

Interpreting expression data

In a study, a tumour suppressor gene shows relative expression of 100 units in healthy tissue but 15 units in cancerous tissue, with no change in its DNA sequence. Interpret this result.

Describe the data
Expression falls from 100 to 15 units - an 85% reduction - in the cancerous tissue.
Explain biologically
As the base sequence is unchanged, the silencing is likely epigenetic: increased methylation of the gene's DNA (or reduced histone acetylation) has switched it off.
Note the consequence and caution
A silenced tumour suppressor gene removes a brake on division, consistent with cancer; but a single correlation does not prove cause, and controls and repeats would be needed.

Result: The tumour suppressor gene is epigenetically silenced (reduced expression despite an unchanged sequence), consistent with loss of cell-cycle control.

Typical mistakes

Assuming a correlation in expression data proves a cause without considering controls, sample size or other variables.
Forgetting that epigenetic silencing (not only mutation) can inactivate a tumour suppressor gene.

Active revision

Data show that a tumour suppressor gene is much less expressed in cancerous tissue than in healthy tissue, but its base sequence is unchanged. Suggest and explain a reason for the reduced expression.

Active recall

Recall the key points — then reveal.

Sources: GCE AS and A level subject content for the sciences (Department for Education) · AQA A-level Biology 7402 specification (AQA)

Recombinant DNA technology and the polymerase chain reaction#

AdvancedAQA 7402 3.8.4.1DfE GCE Biology - recombinant DNA technology

The polymerase chain reaction

Fig. 6Each PCR cycle doubles the DNA: n cycles give 2 to the power n copies per starting molecule.

Recombinant DNA technology transfers a gene (fragment of DNA) from one organism into another, often into a different species, and works because the genetic code is universal - the transferred gene is transcribed and translated in the same way in the new host. The organism that then expresses the gene is transgenic, and the technique is used, for example, to make bacteria produce human insulin. The process has three broad stages: isolating the gene, inserting it into a vector, and getting it into and expressed by host cells.

A DNA fragment (gene) can be obtained in several ways. Reverse transcriptase can make a DNA copy (complementary DNA) from the mRNA of a gene, which is useful because mRNA of a highly expressed gene is plentiful and the cDNA has no introns. Restriction endonucleases are enzymes that cut DNA at specific base sequences (recognition sites); many leave staggered cuts with short single-stranded sticky ends that make it easy to join fragments. A gene machine can also synthesise a short DNA sequence directly from its known base sequence.

The isolated gene is joined into a vector, usually a plasmid (a small circular piece of bacterial DNA). The same restriction endonuclease is used to cut the plasmid, giving complementary sticky ends, and the enzyme DNA ligase joins the gene into the plasmid, forming recombinant DNA. The recombinant plasmid is then taken up by host cells (for example, bacteria are made more permeable so they take up the plasmid). Marker genes (for example, for antibiotic resistance or fluorescence) are included so that the cells which have taken up the plasmid can be identified.

Alternatively, a gene can be amplified directly in vitro (outside a living cell) by the polymerase chain reaction (PCR), an automated cycle that doubles the amount of DNA each round. Each cycle has three steps: denaturation (heating to about 95 degrees C to separate the two DNA strands by breaking the hydrogen bonds); annealing (cooling to about 55 degrees C so that short primers bind to the ends of the target sequence); and extension (heating to about 72 degrees C so that a heat-stable DNA polymerase, Taq polymerase, builds new complementary strands). Because each cycle doubles the DNA,

n

cycles produce

2^n

copies from each starting molecule.

\text{copies} = N_0 \times 2^n

PCR amplification

$N_0$ = starting number of DNA molecules, $n$ = number of cycles; the amount doubles each cycle.

In vivo gene cloning

Fig. 7The gene is joined into a plasmid vector by DNA ligase and taken up by host bacteria.

Worked example

PCR amplification

A PCR reaction starts with 5 copies of a DNA fragment and runs for 10 cycles. Calculate the number of copies produced, assuming the amount doubles each cycle.

Amplification factor
Each cycle doubles the DNA, so 10 cycles multiply the amount by $2^{10} = 1024$ .
Apply the starting number
Copies = $N_0 \times 2^n = 5 \times 1024$ .
$5 \times 2^{10} = 5 \times 1024 = 5120$

Result: 5120 copies after 10 cycles.

Typical mistakes

Confusing the roles of restriction endonuclease (cuts DNA) and DNA ligase (joins DNA).
Forgetting that the same restriction enzyme must be used on the gene and the vector so their sticky ends are complementary.

Active revision

Starting with a single molecule of DNA, calculate the number of copies present after 20 complete cycles of PCR.

Active recall

Recall the key points — then reveal.

Sources: GCE AS and A level subject content for the sciences (Department for Education) · AQA A-level Biology 7402 specification (AQA)

Studying genomes: probes, electrophoresis and genetic fingerprinting#

AdvancedAQA 7402 3.8.4.2AQA 7402 3.8.4.3DfE GCE Biology - gene technologies

Gel electrophoresis

Fig. 8DNA moves to the positive electrode; smaller fragments travel further, separating the fragments by size.

A DNA probe is a short, single-stranded piece of DNA with a base sequence complementary to a target sequence (such as an allele that causes a genetic disease), labelled so that it can be detected (with a radioactive or fluorescent label). When mixed with single-stranded sample DNA, the probe binds (hybridises) to any complementary sequence present; detecting the label then shows whether the target sequence is there. This underlies genetic screening - testing an individual for disease alleles - and medical diagnosis.

Gel electrophoresis separates fragments of DNA by size. The DNA samples are placed in wells at one end of a gel and an electric field is applied; because DNA is negatively charged (its phosphate groups), the fragments move towards the positive electrode. Smaller fragments move through the gel mesh more easily and therefore travel further, so the fragments are separated into bands according to length - the smallest at the far end. Comparing the resulting pattern of bands is the basis of genetic fingerprinting.

Genetic (DNA) fingerprinting uses the non-coding, repetitive regions of the genome called variable number tandem repeats (VNTRs), short base sequences repeated a number of times that varies greatly between individuals. The DNA is cut, amplified and separated by electrophoresis to give a pattern of bands unique to each individual (except identical twins). Because the pattern is inherited, it is used in forensic science (matching a suspect to a sample), in paternity testing and in studying the genetic relationships between organisms.

Reading the whole genome, and the tools above, open up powerful applications that are examined with their benefits and risks. Genetic screening and prenatal testing can identify disease alleles, informing decisions but raising questions of privacy, consent and discrimination (for example, by insurers). Gene therapy aims to treat a genetic disorder by supplying a working allele - somatic therapy treats body cells (its effects are not inherited), whereas germ-line therapy would affect the gametes and future generations, which raises particular ethical concern. A strong answer weighs the medical benefits against these ethical and practical issues.

Worked example

Reading an electrophoresis gel

After electrophoresis, three DNA fragments of 500, 1500 and 4000 base pairs are loaded together in one well. State the order of the bands from the well and explain.

Charge and direction
All the fragments are negatively charged and move towards the positive electrode, away from the well.
Size and distance
Smaller fragments move more easily through the gel mesh and travel further, so the 500 bp fragment moves furthest and the 4000 bp fragment least.
Order
From the well outwards: 4000 bp (nearest), then 1500 bp, then 500 bp (furthest).

Result: From the well: 4000 bp, then 1500 bp, then 500 bp (smallest travels furthest).

Typical mistakes

Saying larger fragments travel further - smaller fragments move more easily through the gel and travel further.
Confusing somatic gene therapy (body cells, not inherited) with germ-line therapy (gametes, inherited).

Active revision

Explain how gel electrophoresis and a labelled DNA probe could be used together to test whether a person carries a particular disease-causing allele.

Active recall

Recall the key points — then reveal.

Sources: GCE AS and A level subject content for the sciences (Department for Education) · AQA A-level Biology 7402 specification (AQA)

References & sources

Sources

Department for Education

GCE AS and A level subject content for the sciences

AQA

AQA A-level Biology 7402 specification

The control of gene expression

6 sections~20 min reading time3 competenciesLevel Standard 1 · Advanced 5

The control of gene expression

Key points

Comparing a substitution and a deletion

Key points

Classifying a stem cell

Key points

Silencing a gene epigenetically

Key points

Interpreting expression data

Key points

PCR amplification

Key points

Reading an electrophoresis gel

Sources

The control of gene expression

Key points

Comparing a substitution and a deletion

Key points

Classifying a stem cell

Key points

Silencing a gene epigenetically

Key points

Interpreting expression data

Key points

PCR amplification

Key points

Reading an electrophoresis gel

Sources