AQA A-Level Biology: Topic 8 The control of gene expression

Gene Mutation and Mutagenic Agents

A gene mutation is a change in the base sequence of DNA arising spontaneously during DNA replication; a mutagenic agent is a factor that increases mutation rate (e.g. UV light, alpha particles).

How a Mutation Leads to a Non-Functional Protein or Enzyme

Changes sequence of DNA base triplets and mRNA codons, changing the amino acid sequence of the polypeptide; this alters the position of hydrogen, ionic, and disulphide bonds, changing the protein's tertiary structure and active site shape so the substrate cannot bind.

Types of Gene Mutations

Substitution: one base replaced by another; Addition: one or more bases added; Deletion: one or more bases lost; Duplication: a sequence of bases repeated; Inversion: a sequence detaches and rejoins in reverse order; Translocation: a sequence detaches and inserts into another location on the same or a different chromosome.

Why Not All Mutations Affect Amino Acid Sequence or Harm the Organism

Substitutions may code for the same amino acid due to the degenerate genetic code, or occur in non-coding introns; even if amino acids change, tertiary structure may stay the same if R-group bonds do not shift, or the change may give a selective advantage.

Frameshift Mutations

Occurs when bases are added, deleted, duplicated, or translocated by a number not divisible by 3, shifting the reading frame so all downstream triplets/codons and amino acids change (or creating an early stop codon).

Stem Cells and Cell Specialisation

Undifferentiated cells able to divide indefinitely by mitosis and differentiate into specialised cells; stimuli activate transcription factors for specific genes, so only those genes are transcribed into mRNA and translated into proteins that permanently modify cell structure and function.

Totipotent, Pluripotent, Multipotent, and Unipotent Stem Cells

Totipotent (early embryos): differentiate into any cell including placenta; Pluripotent (embryos): differentiate into all body cells except placenta; Multipotent (mature mammals, e.g. bone marrow): differentiate into a limited range of cells; Unipotent (mature mammals, e.g. heart cardiomyocytes): differentiate into one cell type only.

Use of Stem Cells in Treating Human Disorders

Transplanted stem cells divide indefinitely and differentiate into healthy cells to replace damaged ones (e.g. islet cells for Type 1 diabetes, or bone marrow transplants after destroying faulty marrow for blood cancers/sickle cell disease).

Producing Induced Pluripotent Stem (iPS) Cells

Obtain adult somatic cells from the patient, add specific protein transcription factors associated with pluripotency so they bind promoter regions and activate pluripotency genes, and culture cells to divide by mitosis.

Evaluating Stem Cell Use in Medicine

For: relieves suffering, uses leftover IVF embryos, and iPS cells avoid embryo destruction and immune rejection; Against: embryonic stem cells destroy a potential life (cannot consent), donor cells risk immune rejection, and stem cells could divide uncontrollably to form tumours.

Transcription Factors and Regulation of Transcription

Proteins in eukaryotes that move from cytoplasm to nucleus and bind specific DNA base sequences on promoter regions upstream of target genes, stimulating or inhibiting transcription by helping or preventing RNA polymerase binding.

How Oestrogen Affects Transcription

Lipid-soluble oestrogen diffuses across the cell membrane and binds its cytoplasmic receptor (an inactive transcription factor), changing its shape into an active oestrogen-receptor complex; this enters the nucleus and binds a specific promoter sequence, helping RNA polymerase bind to stimulate transcription in target cells with receptors.

Epigenetics and the Epigenome

Epigenetics is heritable changes in gene expression without altering the DNA base sequence, caused by environmental factors (e.g. diet, stress, toxins); the epigenome is all chemical modifications to DNA (methyl groups) and histones (acetyl groups).

How DNA Methylation and Histone Acetylation Inhibit Transcription

Increased DNA methylation (adding methyl groups to cytosine) causes nucleosomes to pack tightly; decreased histone acetylation increases histones' positive charge so they bind negatively charged DNA more tightly; both prevent transcription factors and RNA polymerase from binding to promoters.

RNA Interference (RNAi)

Single-stranded siRNA or miRNA combines with a protein to form an RNA-induced silencing complex (RISC), which binds complementary bases on target mRNA and either hydrolyses the mRNA into fragments or blocks ribosome binding, preventing translation.

Benign vs Malignant Tumours

Benign: slow-growing, well-differentiated cells, normal nuclei, encapsulated with clear borders, do not spread by metastasis, rarely return after surgery. Malignant: fast-growing, poorly differentiated, irregular dark nuclei, unencapsulated and invade nearby tissues, spread via blood/lymph (metastasis) due to lack of adhesion molecules, often return.

Tumour Suppressor Genes and Cancer

Code for proteins that slow the cell cycle or trigger apoptosis when DNA is damaged; DNA mutations (making non-functional proteins) or hypermethylation/hypoacetylation (silencing transcription) remove this inhibition, causing uncontrolled cell division (requires mutations in both alleles).

Proto-Oncogenes, Oncogenes, and Cancer

Proto-oncogenes code for proteins that stimulate cell division; mutations (making permanently active proteins) or hypomethylation/hyperacetylation (overproducing proteins) turn them into oncogenes that permanently stimulate uncontrolled cell division (requires mutation in only one allele).

Role of Oestrogen and Drugs in Breast Cancer

High oestrogen forms more active oestrogen-receptor transcription factor complexes that bind promoters of cell-division genes, stimulating transcription and uncontrolled division; drugs shaped like oestrogen bind and block oestrogen receptors so fewer transcription factors bind promoters.

Genome Sequencing and Why Genome Does Not Equal Proteome

Genome sequencing determines an organism's entire DNA base sequence (automated, fast, and cost-effective) to identify proteins/antigens for vaccines, disease alleles, and evolutionary links; the genome cannot directly predict the proteome in eukaryotes due to non-coding introns and regulatory genes.

Why Transferred DNA Works in Transgenic Organisms

Recombinant DNA transferred between species can be transcribed and translated because the genetic code is universal (same triplets code for the same amino acids) and transcription/translation mechanisms are universal.

Producing DNA Fragments Using Restriction Enzymes

Restriction enzymes cut DNA at specific base recognition sequences complementary to their active site on either side of the desired gene, often making a staggered cut that leaves single-stranded sticky ends.

Producing DNA Fragments From mRNA and Gene Machines

From mRNA: isolate mRNA from cells making the protein, use reverse transcriptase with DNA nucleotides to make single-stranded cDNA, then use DNA polymerase to form double-stranded DNA. Gene machine: synthesises intron-free DNA from scratch using amino acid sequences; both methods lack introns so prokaryotes can translate them.

Polymerase Chain Reaction (PCR) Steps

1. Heat to 95°C to break hydrogen bonds and separate DNA strands; 2. Cool to 55°C so forward and reverse primers bind complementary DNA ends; 3. Heat to 72°C so thermostable Taq DNA polymerase joins adjacent DNA nucleotides with phosphodiester bonds (doubles DNA per cycle until primers/nucleotides run out).

Role of Primers in PCR

Short, single-stranded DNA fragments complementary to base sequences at the edges of the target region; required to provide a starting point for DNA polymerase to bind and add nucleotides.

In Vivo DNA Amplification Steps

1. Add promoter (for RNA polymerase binding) and terminator (to stop transcription) regions to DNA fragment; 2. Cut vector (e.g. plasmid) with same restriction enzyme for complementary sticky ends and join with DNA ligase (phosphodiester bonds); 3. Transform host bacteria (e.g. heat shock in Ca2+); 4. Identify transformed cells via marker genes (antibiotic resistance or fluorescence); 5. Culture clones.

Applications of Recombinant DNA Technology

Medicine: GM bacteria make human insulin (ethical, no allergies) and pharming produces cheaper drugs; Agriculture: herbicide-resistant, pest-resistant, or nutrient-enriched crops (Golden Rice); Industry: GM bacteria produce industrial enzymes.

Gene Therapy and Its Limitations

Introducing functional alleles into somatic cells to treat genetic disorders like cystic fibrosis (not heritable; germline therapy is illegal); limited because modified cells have short lifespans requiring repeated treatment, vectors trigger immune responses, and random DNA insertion may disrupt genes and cause cancer.

Evaluating Recombinant DNA Technology

Humanitarians support higher crop yields (reducing famine) and cheaper medicines/gene therapy; environmentalists and anti-globalisation activists oppose potential herbicide-resistant superweeds, reduced food-web biodiversity, and large biotech corporations holding patents.

DNA Probes and DNA Hybridisation

DNA probes are short, single-stranded DNA sequences complementary to a target allele and labelled with a fluorescent or radioactive tag (made long enough to be unique in the genome); DNA hybridisation is the binding of a single-stranded probe to a complementary target DNA strand via hydrogen bonds.

Gel Electrophoresis Principles

Separates DNA/RNA fragments or proteins by length/mass and charge; DNA is negatively charged (due to phosphate groups) and moves through a porous gel in buffer towards the positive electrode, with shorter fragments travelling faster and further (compared against a known standard).

Genetic Screening Using DNA Probes

1. Extract DNA and amplify by PCR; 2. Cut with restriction enzymes; 3. Separate fragments by gel electrophoresis; 4. Transfer to nylon membrane and separate into single strands; 5. Add labelled DNA probes to hybridise with target alleles and wash off unbound probes; 6. Visualise under UV light (fluorescent) or autoradiography/X-ray film (radioactive).

Uses of DNA Probes, Genetic Counselling, and Personalised Medicine

Probes screen for heritable disorders, disease risks, and drug responses; genetic counsellors explain screening results, treatments, preventative lifestyle choices, and inheritance probabilities; personalised medicine tailors drugs to a patient's genotype for higher effectiveness.

Variable Number Tandem Repeats (VNTRs) and Genetic Fingerprinting

VNTRs are repeating base sequences in non-coding DNA whose lengths at different loci vary uniquely between individuals (except identical twins); genetic fingerprinting uses PCR, restriction enzymes, gel electrophoresis, nylon transfer, and labelled DNA probes to compare VNTR band patterns.

Applications of Genetic Fingerprinting

Forensics: matching suspect VNTR bands to crime scene DNA; Paternity and relationships: children share ~50% of VNTR bands with each parent and closer relatives have more similar patterns; Medicine: identifying VNTR patterns linked to disorders like Huntington's; Breeding: avoiding inbreeding by mating individuals with dissimilar fingerprints.