Epigenetics (Methylation & Acetylation), Transcription Factors, siRNA, PCR & Gel Electrophoresis

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Transcription Factors & Oestrogen Mechanism

Transcription factors are intracellular proteins with a specific DNA-binding domain that bind to promoter regions upstream of target genes, either stimulating (activators) or blocking (repressors) the binding of RNA polymerase. Oestrogen is a lipid-soluble steroid hormone: 1. Diffuses directly across the phospholipid bilayer into the cytoplasm/nucleus. 2. Binds to a complementary receptor site on an inactive transcription factor molecule. 3. Binding causes a conformational change in the tertiary structure of the DNA-binding site, activating the transcription factor. 4. The oestrogen-transcription factor complex enters the nucleus through a nuclear pore and binds to a specific base sequence on the DNA promoter, stimulating RNA polymerase to transcribe the target gene into mRNA.

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Epigenetics: DNA Methylation vs Histone Acetylation

Epigenetics is the heritable control of gene expression caused by environmental chemical modifications to DNA and histones that do not alter the underlying DNA base sequence. 1. Increased DNA Methylation (adding -CH₃ groups to cytosine bases in CpG sites): inhibits transcription by preventing transcription factors and RNA polymerase from binding to the promoter and attracting proteins that condense the chromatin (heterochromatin). 2. Decreased Histone Acetylation (removal of acetyl -COCH₃ groups by HDAC enzymes): increases the positive charge on histone tails so they bind more strongly to the negatively charged phosphate groups of DNA; chromatin becomes tightly coiled/condensed so RNA polymerase cannot access the gene (transcription switched off).

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RNA Interference (siRNA & miRNA)

Post-transcriptional gene silencing in the cytoplasm: 1. Double-stranded RNA is cut into short fragments (~21-23 base pairs) called small interfering RNA (siRNA) by the enzyme Dicer. 2. One strand of the siRNA associates with an intracellular enzyme complex called RISC (RNA-induced silencing complex) and unwinds; the single passenger strand is degraded. 3. The remaining single guide strand of siRNA directs the RISC complex to a target mRNA molecule via complementary base pairing. 4. The endonuclease enzyme within RISC hydrolyses (cuts) the phosphodiester backbone of the target mRNA into fragments, preventing ribosomes from translating it into a polypeptide.

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Oncogenes vs Tumour Suppressor Genes in Cancer

Proto-oncogenes code for proteins that stimulate normal cell division (mitosis); a gain-of-function mutation or hypomethylation (under-methylation) converts a proto-oncogene into a permanently active oncogene, causing uncontrolled cell division. Tumour suppressor genes (e.g. TP53, BRCA1) code for proteins that slow cell division, repair DNA damage, or trigger apoptosis; loss-of-function mutations or hypermethylation (excess methylation of the promoter) silence both alleles of a tumour suppressor gene, removing the brakes on the cell cycle.

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Polymerase Chain Reaction (PCR) Steps & Components

In vitro amplification of target DNA fragments using DNA template, free dNTP nucleotides, short single-stranded DNA primers, Mg²⁺ cofactor ions, and thermostable Taq DNA polymerase (from Thermus aquaticus bacteria, which does not denature at 95°C): 1. Denaturation (95°C): breaks hydrogen bonds between complementary DNA strands to separate them into single strands. 2. Annealing (55°C): cools so DNA primers bind (hydrogen bond) to complementary sequences at the 3' ends of each flanking target strand (provides a 3'-OH starting point for DNA polymerase and prevents the two template strands from simply re-annealing). 3. Extension/Synthesis (72°C): optimum temperature for Taq polymerase to add complementary dNTPs in the 5'→3' direction. Doubles the number of DNA copies each cycle (2ⁿ copies after n cycles).

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DNA Probes, VNTRs & Gel Electrophoresis

DNA probes are short, single-stranded DNA sequences labelled with a fluorescent dye or radioactive isotope (³²P) that hybridise by complementary base pairing to screen for specific mutant alleles on a DNA microarray. Genetic fingerprinting analyses Variable Number Tandem Repeats (VNTRs / minisatellites): non-coding repetitive sequences whose length/repeat number is unique to individuals (except identical twins) and inherited from both parents. Restriction endonucleases cut DNA outside the VNTRs; fragments are loaded into agarose gel wells and an electric current is applied: negatively charged DNA (due to PO₄³⁻ groups) moves toward the positive anode, with shorter/smaller fragments moving faster and further through the gel pores.

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1. Q1

   Explain how oestrogen stimulates the transcription of a target gene in a human breast tissue cell, and explain why oestrogen has no effect on cells that lack the specific oestrogen receptor. (5 marks)

   Check Mark Scheme

   Click “Check Mark Scheme” to compare your answer, 1. Because oestrogen is a lipid-soluble steroid hormone, it diffuses directly across the phospholipid bilayer of the target cell surface membrane [1 mark]. 2. Inside the cell, oestrogen binds to a complementary receptor site on an inactive transcription factor protein [1 mark]. 3. Binding causes a conformational change in the tertiary structure of the transcription factor's DNA-binding site, activating it [1 mark]. 4. The oestrogen-transcription factor complex diffuses into the nucleus via a nuclear pore and binds to a specific base sequence on the promoter region upstream of the target gene, stimulating RNA polymerase to bind and transcribe the gene into mRNA [1 mark]. 5. Cells lacking the complementary oestrogen receptor cannot form the active transcription factor complex, so RNA polymerase is not recruited to the gene promoter [1 mark].

2. Q2

   Explain how hypermethylation of a tumour suppressor gene and hypomethylation of a proto-oncogene can both contribute to the development of a malignant tumour. (5 marks)

   Check Mark Scheme

   Click “Check Mark Scheme” to compare your answer, 1. Hypermethylation involves adding excess methyl (-CH₃) groups to cytosine bases in the CpG promoter region of a tumour suppressor gene [1 mark]. 2. This prevents transcription factors and RNA polymerase from binding to the promoter and condenses the DNA-histone complex, silencing transcription of the tumour suppressor gene [1 mark]. 3. Because the tumour suppressor protein (which normally slows mitosis or triggers apoptosis) is not produced, cell division proceeds unchecked [1 mark]. 4. Conversely, hypomethylation (loss of methyl groups) of a proto-oncogene leaves the chromatin loosely packed (euchromatin) and accessible to transcription factors and RNA polymerase [1 mark]. 5. The proto-oncogene acts as an oncogene and is over-transcribed, producing excessive growth-stimulating proteins that drive rapid, uncontrolled mitotic cell division to form a tumour [1 mark].

3. Q3

   A forensic scientist found a tiny trace of blood containing 10 copies of a suspect's genomic DNA at a crime scene. Calculate how many copies of a target VNTR locus would be present after 20 cycles of PCR, and explain how the amplified DNA is used to produce a genetic fingerprint that can be compared to suspects. (5 marks)

   Check Mark Scheme

   Click “Check Mark Scheme” to compare your answer, 1. Number of copies = Initial copies × 2ⁿ = 10 × 2²⁰ = 10 × 1,048,576 = 1.05 × 10⁷ copies (10,485,760 copies) [1 mark]. 2. Restriction endonucleases are used to cut the DNA at specific palindromic recognition sites flanking the VNTR regions (or PCR primers amplify the VNTRs directly) [1 mark]. 3. The DNA fragments are pipetted into wells in an agarose gel and a direct electric current is applied; negatively charged DNA fragments migrate toward the positive anode, with shorter VNTR fragments moving faster and further than longer ones [1 mark]. 4. Single-stranded radioactive or fluorescently labelled DNA probes complementary to the VNTR core repeats are added (after transferring to a nylon membrane in Southern blotting) so they hybridise by complementary base pairing [1 mark]. 5. Exposure to X-ray film or UV light reveals a pattern of dark or glowing bands; if all bands from the crime scene sample match the exact positions of a suspect's bands on the same gel, the DNA came from that suspect [1 mark].

4. Q4

   Compare and contrast in vivo gene cloning (using transformed bacteria) and in vitro gene amplification (using the Polymerase Chain Reaction). (4 marks)

   Check Mark Scheme

   Click “Check Mark Scheme” to compare your answer, 1. Speed and automation: PCR is much faster (producing billions of copies in a few hours in an automated thermocycler), whereas in vivo cloning requires days to cut plasmids, ligate DNA, transform bacteria, and culture colonies on agar plates [1 mark]. 2. Error rate: In vivo bacterial DNA replication includes proofreading enzymes so it has a very low mutation/error rate, whereas Taq polymerase lacks 3'→5' proofreading activity and introduces more base-copying errors [1 mark]. 3. Protein expression: In vivo cloning inside living bacteria can transcribe and translate the inserted gene directly into a functional human protein (e.g. insulin) and replicate very large DNA fragments, whereas PCR only produces naked DNA copies and is limited to relatively short fragments (<10 kb) [1 mark]. 4. Contamination sensitivity: PCR amplifies any contaminating DNA molecule exponentially, whereas in vivo cloning uses restriction enzymes and selectable antibiotic-resistance markers to isolate pure recombinant clones [1 mark].

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