Topic 8.2-8.4

What are transcription factors?

Proteins that regulate transcription of specific target genes in eukaryotes by binding to specific DNA sequences on promoter regions.

How do transcription factors regulate transcription?

• Move from cytoplasm to nucleus

• Bind to a promoter region on DNA

• Stimulate or inhibit transcription by affecting RNA polymerase binding

How does oestrogen affect transcription?

1. Diffuses into cell (lipid-soluble)

2. Binds to its receptor (inactive TF) forming an oestrogen-receptor complex

3. Complex becomes an active TF

4. Moves to nucleus and binds promoter region

5. Stimulates transcription by helping RNA polymerase bind

Why does oestrogen only affect target cells?

Only target cells have oestrogen receptors.

What is epigenetics?

Heritable changes in gene expression without changes to the DNA base sequence, often influenced by environmental factors.

What is the epigenome?

All chemical modifications to DNA and histones, including:

• Methyl groups on DNA

• Acetyl groups on histones

How does methylation and acetylation allow transcription?

↓ Methylation of DNA
↑ Acetylation of histones

How does methylation and acetylation inhibit transcription?

↑ Methylation of DNA
↓ Acetylation of histones

How does increased methylation inhibit transcription?

• Methyl groups added to cytosine bases

• Nucleosomes pack tightly

• Transcription factors & RNA polymerase can't bind to promoter

How does decreased acetylation inhibit transcription?

• Increases histone positive charge

• DNA binds more tightly to histones

• Transcription factors & RNA polymerase can't bind

How can epigenetics influence disease and treatment?

• Environmental factors can cause harmful epigenetic changes

• These may increase or inhibit gene expression → disease

• Epigenetic markers help in early diagnosis

• Drugs can reverse methylation/acetylation abnormalities

What is RNA interference (RNAi)?

Inhibition of mRNA translation by RNA molecules like siRNA or miRNA, silencing target genes.

How does RNAi regulate translation?

• siRNA or miRNA + protein → RNA-induced silencing complex (RISC)

• Binds complementary target mRNA

• Causes mRNA hydrolysis or blocks ribosome binding

• Prevents translation into protein

How is siRNA different from miRNA?

• siRNA: Synthesised as double-stranded RNA; one strand is used in RISC

• miRNA: Formed as a hairpin loop; both strands used in RISC

How do tumours and cancers form?

Mutations in DNA/genes controlling mitosis cause uncontrolled cell division. A mass of abnormal cells forms a tumour.

What is the difference between malignant and benign tumours?

• Malignant: Cancerous, can spread (metastasise)

• Benign: Non-cancerous, doesn’t spread

Compare benign and malignant tumours in terms of growth.

• Benign: Grow slowly

• Malignant: Grow quickly

Compare the differentiation of cells in benign and malignant tumours.

• Benign: Well differentiated

• Malignant: Poorly differentiated

Do benign tumours invade other tissues?

No, they have defined borders and are usually encapsulated.

Do malignant tumours invade other tissues?

Yes, they have poorly defined borders and can spread to other tissues.

How do malignant tumours spread?

Through metastasis—cells break off and travel to other parts of the body due to lack of adhesion molecules.

What is the function of tumour suppressor genes?

Code for proteins that inhibit the cell cycle or cause apoptosis of abnormal cells.

How do mutations in tumour suppressor genes lead to tumours?

• Non-functional protein produced

• Epigenetic silencing (↑ methylation or ↓ acetylation)
  → Cell division can't be controlled

What is the function of proto-oncogenes?

Code for proteins that stimulate cell division (e.g. in response to growth factors).

How do oncogenes lead to tumour formation?

• Mutation causes overactive or overproduced protein

• Epigenetic changes (↓ methylation or ↑ acetylation) → increased transcription
  → Uncontrolled cell division

Why must both tumour suppressor gene alleles be mutated, but only one proto-oncogene allele?

• One normal TSG allele still produces enough protein

• One mutated oncogene allele is enough to stimulate excessive division

How is epigenetics relevant to cancer treatment?

Drugs can reverse epigenetic changes:

• ↑ Methylation or ↓ Acetylation of oncogenes (↓ expression)

• ↓ Methylation or ↑ Acetylation of TSGs (↑ expression)

How does oestrogen promote some breast cancers?

• Binds to oestrogen receptors (inactive TFs)

• Activates them → forms oestrogen-receptor complexes

• These bind DNA promoter regions → increase transcription of cell cycle genes

• Stimulates uncontrolled cell division

How do oestrogen-like drugs treat oestrogen receptor-positive breast cancers?

• Bind to oestrogen receptors, blocking oestrogen

• Prevent transcription of genes that stimulate cell division

What is the genome?

The complete set of genes in a cell.

What is the proteome?

The full range of proteins a cell can produce (coded for by the cell’s DNA/genome).

What is genome sequencing?

Identifying the DNA base sequence of an organism’s genome.

Why is genome sequencing important?

Allows determination of the amino acid sequences of proteins from genetic code.

How can sequencing the genome of a pathogen help develop vaccines?

• Identifies the pathogen’s proteome

• Allows identification of antigens to use in vaccines

What are some other potential applications of genome sequencing?

• Identify genes/alleles for genetic diseases and cancers

• Develop targeted drugs or gene therapy

• Screen patients for early prevention/personalised medicine

• Identify species and determine evolutionary relationships

Why can’t the genome be directly translated into the proteome in complex organisms?

• Presence of non-coding DNA (e.g. introns)

• Presence of regulatory genes (e.g. those coding for miRNA)

How have sequencing methods changed over time?

• Now automated

• Faster, cheaper, and scalable

• Continuously improving

What is recombinant DNA technology?

Transfer of DNA fragments from one organism or species to another.

Why can transferred DNA be translated within recipient cells?

Because the genetic code and transcription/translation mechanisms are universal.

How are DNA fragments produced using restriction enzymes?

1. Cut DNA at specific recognition sequences

2. Often form 'sticky ends' with single-stranded overhangs

How are DNA fragments produced from mRNA?

1. Isolate mRNA from a cell

2. Use reverse transcriptase to make cDNA

3. Use DNA polymerase to synthesize second DNA strand

Two advantages of obtaining genes from mRNA over DNA?

• mRNA is more abundant in protein-synthesizing cells

• mRNA lacks introns, so easier for prokaryotic expression

How are DNA fragments produced using a gene machine?

Synthesizes DNA from known amino acid sequences without templates; no introns present.

Name one in vitro and one in vivo amplification technique.

• In vitro: Polymerase Chain Reaction (PCR)

• In vivo: Culturing transformed host cells

Steps of PCR?

1. 95°C: DNA strands separate

2. 55°C: Primers bind

3. 72°C: DNA polymerase builds new strands

Role of primers in PCR?

Allow DNA polymerase to start synthesis; complementary to target DNA ends

Why does PCR eventually stop?

Primers and nucleotides are used up.

Summarize the in vivo amplification of DNA fragments.

1. Add promoter/terminator regions

2. Insert into vectors with marker genes

3. Transform host cells

4. Detect GM cells using marker genes

5. Culture transformed cells

Why add promoter and terminator regions to DNA fragments?

• Promoters allow RNA polymerase binding and start transcription (can target specific cell types)

• Terminators signal transcription to stop.

What is the role of vectors in recombinant DNA technology?

Transfer DNA into host cells or organisms (e.g., plasmids, viruses).

How do enzymes insert DNA fragments into vectors?

• Restriction enzymes cut vector DNA creating sticky ends complementary to DNA fragment ends

• DNA ligase joins DNA fragment to vector by forming phosphodiester bonds

How are host cells transformed using vectors?

• Plasmids enter cells (e.g., heat shock with calcium ions)

• Viruses inject DNA that integrates into host DNA

Why are marker genes inserted into vectors?

To detect transformed cells, e.g., antibiotic resistance or fluorescent protein genes allow identification.

How is recombinant DNA technology useful in medicine?

• GM bacteria produce human proteins like insulin

• GM animals/plants produce pharmaceuticals (‘pharming’)

• Gene therapy for genetic disorders

What are agricultural uses of recombinant DNA technology?

• GM crops resistant to herbicides or insects

• GM crops with improved nutrition (e.g., Golden Rice)

• GM animals with increased growth hormone

What are industrial applications of recombinant DNA technology?

GM bacteria produce enzymes used in industry and food production.

What is gene therapy?

Introduction of new DNA (healthy alleles) into cells to overcome effects of faulty alleles causing genetic disorders.

What issues are associated with gene therapy?

• Short-lived effect needing repeated treatment

• Immune responses to modified cells or viruses

• Unknown long-term side effects (e.g., cancer risk)

Why might humanitarians support recombinant DNA technology?

• Increased crop yields reduce famine

• Gene therapy can cure genetic disorders

• Pharming makes medicines more accessible and affordable

Why might environmentalists or anti-globalisation activists oppose recombinant DNA technology?

• Risk of ‘superweeds’ from gene transfer

• Negative effects on ecosystems and biodiversity

• Control of technology by large companies

What are DNA probes?

Short, single-stranded DNA pieces with a base sequence complementary to a target allele region, labelled with a fluorescent or radioactive tag for identification.

Why are DNA probes longer than just a few bases?

Short sequences occur many times in the genome; longer sequences are more specific to the target allele.

What is DNA hybridisation?

Binding of a single-stranded DNA probe to a complementary single strand of DNA by forming hydrogen bonds/base pairs.

How can genetic screening locate specific alleles of genes?

• Extract and amplify DNA by PCR

• Cut DNA with restriction enzymes around target gene

• Separate fragments by gel electrophoresis

• Transfer to nylon membrane and denature to single strands

• Add labelled DNA probes that hybridise with target alleles

• Detect bound probes using UV light or autoradiography

What is gel electrophoresis?

A technique that separates nucleic acid or protein fragments based on length/mass and charge.

How does gel electrophoresis separate DNA fragments?

DNA samples are loaded into a gel, an electric current is applied, and DNA moves towards the positive electrode; shorter fragments travel further.

How can gel electrophoresis results be interpreted?

By comparing unknown DNA fragments to a standard of known fragment lengths; shorter fragments travel further/faster.

Give examples of how labelled DNA probes are used.

• Screening for heritable diseases (e.g., cystic fibrosis)

• Screening drug response alleles

• Screening genetic health risk factors (e.g., predisposition to high cholesterol)

What is the role of a genetic counsellor?

• Explain genetic screening results and disease consequences

• Discuss treatment options

• Advise on lifestyle changes or precautions

• Explain inheritance probabilities to inform reproductive decisions

What is personalised medicine?

Treatment tailored to an individual’s genotype to improve effectiveness, such as targeting specific mutations causing disease.

What are advantages of genetic screening?

• Enables lifestyle changes to reduce disease risk

• Informs reproductive choices

• Allows use of personalised medicines

What are disadvantages of genetic screening?

• Screening for incurable or late-onset diseases can cause depression

• Risk of discrimination by insurers/employers

• May cause undue stress if disease does not develop

What are variable number tandem repeats (VNTRs)?

Repeating sequences of nucleotides (e.g., GATA) found in non-coding DNA at many sites throughout the genome.

Why are VNTRs useful in genetic fingerprinting?

The chance of two individuals having the same VNTRs is very low because many VNTR sites exist and lengths vary at each locus between individuals.

How is genetic fingerprinting used to analyse DNA fragments?

1. Extract and amplify DNA by PCR

2. Cut DNA around VNTRs using restriction enzymes

3. Separate fragments by gel electrophoresis

4. Transfer to nylon membrane and denature to single strands

5. Add labelled DNA probes that hybridise with VNTRs

6. Detect bound probes by UV light or autoradiography

Compare genetic fingerprinting and genetic screening.

• Both amplify DNA with PCR

• Both use electrophoresis to separate DNA fragments

• Both use labelled DNA probes

• Genetic fingerprinting analyses VNTRs; genetic screening analyses specific gene alleles

How can genetic fingerprinting determine genetic relationships?

Closely related organisms share more similar VNTRs; in paternity testing, a child shares about 50% of VNTR bands with the father.

How does genetic fingerprinting assess genetic variability in populations?

More differences in VNTRs indicate greater genetic diversity due to mutations.

How is genetic fingerprinting used in forensic science?

Comparing suspect’s genetic fingerprint to DNA from a crime scene; many matching bands suggest presence at the scene.

How is genetic fingerprinting used in medical diagnosis?

Certain VNTR patterns are linked to increased risk of genetic disorders, like Huntington’s disease.

How is genetic fingerprinting used in animal and plant breeding?

It shows relatedness to avoid inbreeding by breeding individuals with dissimilar genetic fingerprints.