How the base sequence goes wrong

A gene mutation changes the base sequence, and therefore potentially the primary structure, tertiary structure and function of the protein.

• Substitution — one base replaced. Often silent, because the code is degenerate. It can be missense (a different amino acid) or nonsense (a premature stop codon).
• Deletion or insertion (addition) — causes a frameshift: every triplet downstream is read differently, so the protein is almost always non-functional.
• Duplication — a base is repeated (also a frameshift). Inversion — a sequence is reversed. Translocation — a section moves to a different chromosome, which can disrupt genes at both ends.

Mutagenic agents — high-energy ionising radiation, UV light, and chemicals such as benzopyrene in tobacco smoke — increase the rate of mutation. Most mutations are neutral or harmful, but they are the sole ultimate source of new alleles, and therefore of all genetic variation.

Potency and induced pluripotency

All the cells of a multicellular organism contain the same genes; they differ because different genes are expressed. A stem cell is unspecialised and can both self-renew and differentiate.

• Totipotent — can become any cell type, including the extra-embryonic (placental) tissues. Only the zygote and the cells of the very early embryo are totipotent.
• Pluripotent — found in embryos; can become any body cell type, but not placental cells.
• Multipotent — found in adults (e.g. bone marrow); can form a limited number of types, such as the different blood cells.
• Unipotent — can form only one type; for example the cardiomyocytes formed from unipotent stem cells in the heart.

Induced pluripotent stem (iPS) cells are made by taking an adult, fully differentiated body cell and reprogramming it — using transcription factors — so that it becomes pluripotent again. They can then divide indefinitely.

Why iPS cells matter ethically: they can be made from the patient’s own cells, so they are genetically identical and will not be rejected, and no embryo is destroyed. That sidesteps the central objection to embryonic stem cells — although concerns about tumour formation remain.

Potency

Transcription factors and oestrogen

Gene expression is controlled mainly at transcription. A transcription factor is a protein that moves from the cytoplasm into the nucleus and binds to a specific promoter region of DNA, near the start of its target gene. Binding either allows RNA polymerase to bind and begin transcription (activation) or prevents it (repression).

Oestrogen is the exemplar you must know:

• Oestrogen is lipid-soluble, so it diffuses straight through the phospholipid bilayer into the cell.
• Inside, it binds to a specific receptor site on a transcription factor, which is normally held inactive by an inhibitory molecule.
• Binding changes the shape of the receptor, releasing the inhibitor and activating the DNA-binding site. The oestrogen–receptor complex enters the nucleus, binds the promoter, and stimulates transcription of the target gene.

Contrast this with adrenaline (3.6): adrenaline is water-soluble, so it cannot cross the membrane and must act via a surface receptor and the second messenger cAMP. Steroid hormones act on transcription; peptide hormones act through second messengers. Know which is which.

Methylation, acetylation and siRNA

Epigenetics is heritable change in gene expression without any change to the base sequence of the DNA. Environmental factors (diet, stress, toxins) cause chemical tags to be added to the DNA or its associated histones — and those tags can be passed to daughter cells, and sometimes to offspring.

• Increased methylation of DNA — methyl groups are added to cytosine bases in the promoter region. This prevents transcription factors from binding, and attracts proteins that condense the DNA. The gene is switched OFF.
• Decreased acetylation of histones — removing acetyl groups makes the histones more positively charged, so they bind the negatively charged DNA more strongly. The chromatin becomes more condensed, transcription factors cannot reach the gene, and it is switched OFF. (Increased acetylation does the opposite: chromatin loosens, and the gene is switched on.)

siRNA — silencing after transcription: double-stranded RNA is cut by an enzyme into short (~21-base) fragments. One strand of each fragment is loaded into an enzyme complex, which uses it to find an mRNA with a complementary base sequence. The complex then cuts the mRNA into fragments, so it can never be translated. The gene has effectively been silenced.

The therapeutic hook: because epigenetic changes are reversible (unlike a mutation), drugs that inhibit DNA methylation or histone deacetylation can potentially reactivate a silenced tumour suppressor gene. Several are already licensed cancer treatments.

Switching a gene off

How siRNA silences a gene

Oncogenes and tumour suppressor genes

A tumour is the result of uncontrolled mitosis. Two classes of gene normally hold the cell cycle in check, and a mutation in either can release it.

• Proto-oncogenes normally stimulate cell division in response to growth factors. A mutation can turn one into an oncogene that is permanently activated — the receptor may be permanently "on", or excessive growth factor is produced. Division is over-stimulated. This is a gain of function mutation, and one mutated copy is enough.
• Tumour suppressor genes normally slow the cell cycle and trigger apoptosis (programmed cell death) in damaged cells. A mutation, or hypermethylation of the promoter, inactivates the gene, so damaged cells are no longer stopped or destroyed. This is a loss of function.

Benign vs malignant: a benign tumour grows slowly, is often encapsulated, and does not spread. A malignant tumour grows rapidly, has an irregular boundary, and cells break away and spread — metastasis — forming secondary tumours elsewhere.

Oestrogen and breast cancer: after the menopause, fat cells continue to produce oestrogen, and it can accumulate in breast tissue. Oestrogen activates transcription factors that stimulate cell division, so a raised concentration increases the number of divisions and hence the chance of a mutation. This is why the length of oestrogen exposure over a lifetime is a recognised risk factor.

Making recombinant DNA

To get a bacterium to make a human protein such as insulin, you need the gene, a way in, and a way of finding the cells that took it up.

1. Isolating the gene. Three routes:

• Reverse transcriptase on mRNA from a cell that already expresses the gene, giving cDNA. This is the usual choice, because mRNA is abundant in such cells and — crucially — cDNA has no introns. Bacteria cannot splice, so a gene copied straight from human DNA would not be expressed correctly.
• Restriction endonucleases cut the gene out at specific palindromic recognition sequences.
• A gene machine synthesises the sequence from scratch.

2. Inserting it into a vector. The same restriction enzyme cuts both the gene and a plasmid, producing complementary sticky ends — short single-stranded overhangs that base-pair. DNA ligase then joins the sugar-phosphate backbones, forming recombinant DNA.

3. Transformation and identification. The plasmids are mixed with bacteria (with Ca²⁺ ions and heat shock to increase membrane permeability). Only a small proportion take up a plasmid, so marker genes are used: for example a gene for fluorescence, or for antibiotic resistance, so that transformed cells can be identified and grown on.

PCR, electrophoresis and DNA probes

The polymerase chain reaction amplifies DNA in vitro. Each cycle doubles the number of DNA molecules.

• Denaturation, 95 °C — the hydrogen bonds between the two strands break; the DNA separates.
• Annealing, 55–65 °C — primers (short single-stranded DNA) bind to the ends of each target strand. They mark the start point and give DNA polymerase somewhere to attach.
• Extension, 72 °C — Taq polymerase (from a thermophilic bacterium, so it is not denatured at 95 °C) builds the complementary strands from free nucleotides.

Gel electrophoresis: DNA fragments are loaded into wells in an agarose gel and a voltage is applied. DNA is negatively charged (the phosphate groups), so it moves towards the anode (+). Smaller fragments move further, because they pass through the gel more easily. The fragments are therefore separated by length.

DNA probes: a short single-stranded DNA sequence, labelled radioactively or fluorescently, that is complementary to the sequence being looked for. It hybridises (base-pairs) with the target, which can then be detected. Used to screen for alleles associated with genetic disorders or with cancer.

Genetic fingerprinting uses the variable number tandem repeats (VNTRs) in the non-coding DNA between genes. The number of repeats is highly variable between individuals, so the pattern of fragment lengths after electrophoresis is effectively unique (identical twins excepted). Used in forensics, paternity testing, and in determining relatedness in breeding programmes.

Your turn — PCR amplification

Your turn — PCR again

Your turn — restriction fragments

Why cDNA?

Which field does it belong to?

Tap a statement, then tap the area of the topic it belongs to.

Match the potency

Tap an item on the left, then its partner on the right.

The big ideas to take away

That is the whole of AQA 3.8 The control of gene expression. Press Finish to see your score.