8. Gene Expression

What is a gene mutation?

Change in base sequence of DNA

When are gene mutations likely to occur?

Can arise spontaneously during DNA replication, during interphase of cell cycle.

What are the 6 types of gene mutations?

1. Substitution

2. Addition

3. Deletion

4. Inversion

5. Duplication

6. Translocation

What is a substitution mutation and what effect does it have on the primary structure?

- 1 base replaced with another

- No change due to degenerate nature of genetic code

OR

- 1 triplet / codon change → 1 amino acid change

What is an addition mutation and what effect does it have on the primary structure?

- 1 or more bases added to base sequence

- Frameshift; triplets / codons change downstream of mutation → amino acid sequence changes

- If multiple of 3 bases added - no frameshift, but extra triplets / codons → extra amino acids

What is a deletion mutation and what effect does it have on the primary structure?

- 1 or more bases lost from base sequence

- Frameshift; triplets / codons change downstream of mutation → amino acid sequence changes

- If multiple of 3 bases lost - no frameshift, but missing triplets / codons → missing amino acids

What is an inversion mutation and what effect does it have on the primary structure?

- A sequence of bases is separated from DNA and inserted at the same position, backwards

- No frameshift because number of bases stays the same

- Triplets / codons in inverted region change → sequence of amino acids encoded by inverted region change

What is a duplication mutation and what effect does it have on the primary structure?

- A sequence of bases is inserted twice, or multiple times

- Frameshift; triplets / codons change downstream of mutation → amino acid sequence changes

OR

- If multiple of 3 bases added - no frameshift, but extra triplets / codons → extra amino acids

What is a translocation mutation and what effect does it have on the primary structure?

- Sequence of bases taken out and inserted at a different position on the same, or a different chromosome

- Significant impact on gene expression and amino acid sequences at original and new location

What are mutagenic agents?

- Increase the rate of gene mutation (above the rate of naturally occurring mutations)

- Ionising radiation (gamma and X-rays), carcinogens e.g. mustard gas, some viruses

In what instances will a mutation not have an effect on the order of amino acids in the polypeptide?

- Some gene mutations (substitution) change only 1 codon

- New codon might still code for same amino acid because genetic code is degenerate

- Also, some gene mutations occur in the introns and therefore won't affect amino acid sequences

What is frameshift and when does it occur?

- A frameshift occurs when gene mutations such as insertion or deletion change the number of nucleotides by any number not divisible by 3

- This shifts the way the genetic code is read, so all the DNA triplets / mRNA codons downstream from the mutation change

- The sequence of amino acids encoded changes accordingly and the effects on the encoded polypeptide are significant

What are stop codons and how can they be affected by mutations?

- There are 3 stop codons in the genetic code (UAA, UGA, UAG)

- Unlike other codons, these don't code for amino acids, so they terminate translation

- A mutation (substitution or frameshift) may create a premature stop codon and result in the production of a shorter and often non-functional polypeptide

How can mutations lead to the production of a non-functional protein/enzyme? (6)

1. Change in base / triplet sequence of DNA / gene

2. Changes sequence of codons on mRNA

3. Changes sequence of amino acids in primary structure of polypeptide

4. Changes position of hydrogen / ionic / disulphide bonds in protein tertiary structure

5. Changes tertiary structure / shape of protein and in the case of enzymes, the active site will change shape

6. In the case of enzymes, the substrate will be unable to bind to active site and form an enzyme-substrate complex

What are the 4 potencies of stem cells?

1. Totipotency

2. Pluripotency

3. Multipotency

4. Unipotency

What are stem cells?

Unspecialised cells capable of:

- self-renewal; can divide to replace themselves

- specialisation/differentiation; can develop into other types of cell

How does stem cell specialisation occur?

1. Stimulus e.g. chemical

2. Causes selective activation of genes - some genes activated while others inactivated • E.g. muscle cells genes coding for actin and myosin need to be activated

3. mRNA only transcribed from active genes → translated on ribosomes = proteins

4. These proteins modify cell permanently and determine cell structure / function

What are totipotent cells?

- Can divide and differentiate into every cell type in body (including the cells that support the embryo, such as the placenta)

- Occur for a limited time in early mammalian embryos

What are pluripotent cells?

- Can divide and differentiate into most cell types (every cell type in body but not the cells of the placenta)

- Found in embryos

What are multipotent cells?

- Can divide and differentiate into a limited number of cell types e.g. multipotent cells in bone marrow can differentiate into different types of blood cell

- Found in mature mammals

What are unipotent cells?

- Can divide and differentiate into just one cell type e.g. cardiomyocytes (cardiac muscle cells) can be made from unipotent stem cell

- Found in mature mammals

How are stem cells used in medicine?

- Regrow damaged tissues in accidents (i.e. skin grafts) or by disease (i.e. neuro-degenerative diseases, Parkinson's disease)

- B cells of the pancreas in type 1 diabetes

- Drug testing - used to grow artificial tissues

- Developmental biology research - provide insight into embryological development

How are induced pluripotent stem cells produced? (iPS)

1. Produced from adult somatic cells (non-pluripotent cells or fibroblasts)

2. Specific protein transcription factors associated with pluripotency put into cells, causing the cell to express genes associated with pluripotency (reprogrammed)

3. Cells cultured

4. Induced pluripotent stem cells

Why are induced pluripotent stem cells used in medical treatment instead of embryonic cells?

✓ No immune rejection as can be made using patient's own cells

✓ Overcome some ethical issues with using embryonic stem cells e.g. no destruction of embryo and adult can give permission

Evaluate use of stem cells in treating human disorders.

For:

- Use of embryonic stem cells - Tiny ball of cells, incapable of feeling pain, not equivalent to a human

- Would otherwise be destroyed (if from infertility treatment which creates more than needed)

- Duty to apply knowledge to relieve human suffering

Against:

- Use of embryonic stem cells - embryo is a potential human; should be given rights

- Induced pluripotent stem cells - cannot yet reliably reprogramme stem cells

- Could begin to multiply out of control, and cause tumours

What are transcription factors and how do they work?

- Transcription factors are proteins

- Move from cytoplasm → nucleus

- Bind to DNA at a specific DNA base sequence on a promotor region (near start / upstream of target gene)

- Stimulate ('activator') or inhibit ('repressor') transcription (the production of mRNA) of target gene

- By helping or preventing RNA polymerase binding

How does oestrogen initiate transcription?

1. Oestrogen, a steroid hormone, can diffuse across the phospholipid bilayer of the cell-surface membrane as it's lipid soluble.

2. In cytoplasm, oestrogen binds to a receptor of an inactive transcription factor, forming a hormone-receptor complex

3. Inactive transcription factor changes shape, resulting in active transcription factor

4. Diffuses from cytoplasm into nucleus and binds to specific DNA base sequence on a promotor region

5. Stimulates transcription of genes by helping RNA polymerase to bind

What is a nucleosome?

- DNA wrapped around histone proteins

- How closely the DNA and histone are packed together affects transcription

What is epigenetics?

Heritable changes in gene function/expression without changes to the base sequence of DNA, caused by changes in the environment

How does methylation of DNA inhibit transcription?

- Methyl groups added to cytosine bases in DNA

- Nucleosomes pack more tightly together → prevents transcription factors binding; genes not transcribed (RNA polymerase can't bind)

- Irreversible

How does decreased acetylation of associated HISTONES inhibit transcription?

- Decreased acetylation of increases positive charge of histones (think of acetyl groups as caps on positive charge)

- Histones bind DNA (which is negatively charged) more tightly → preventing transcription factors binding; genes not transcribed

- Reversible

How can epigenetics lead to tumour development?

Epigenetic changes that increase the expression of an oncogene, or that silence a tumour suppressor gene, can lead to tumour development

How can epigenetics be used to detect and treat cancer?

- Tests can be used to see if a patient has abnormal levels of methyl and acetyl - early indicator of cancer (called a biomarker)

- Could be manipulated to treat cancer i.e. drugs to prevent histone acetylation / DNA methylation that may have caused these genes to be switched on/off, resulting in cancer

What is RNA interference? (RNAi)

When RNA molecules inhibit translation of mRNA produced by transcription (gene is 'switched on' but encoded protein not produced = 'silenced' gene)

What are the 2 way RNA interference be moderated?

1. Micro-RNA (miRNA) - formed as hair-pin bends of RNA but processed into single-strands 22-26 nucleotides long, both become incorporated into a protein-based RISC (RNA-induced silencing complex)

2. Small interfering RNA (siRNA) - formed as double-stranded molecules 21-25 bp long, one strand incorporated into a protein-based RISC

How do miRNA/siRNA prevent translation?

- Single-stranded miRNA/siRNA within a RISC binds to a molecule of mRNA containing a sequence of bases complementary to its own → mRNA hydrolysed / translation stopped

- miRNA expression deregulated in many human diseases including cancer → offer opportunities as biomarkers and novel therapies

What is the difference between a tumour and cancer?

Tumour - uncontrolled cell division. Not all tumours are cancerous, they can be classified as:

- Benign (non-cancerous; don't spread)

- Malignant (cancerous; spread easily throughout the body via metastasis)

What are the differences between benign and malignant tumours?

- Benign tumours grow slowly (rare mitoses) WHEREAS malignant tumours grow rapidly (more frequent and / or abnormal mitoses)

- Benign tumours are well differentiated / specialised (cells retain function and normal shape, regular nuclei) WHEREAS malignant tumour cells become unspecialised / poorly differentiated

- Benign tumour cells have normal, regular nuclei WHEREAS malignant tumour cells have irregular, larger/darker nuclei

- Benign tumours have well defined borders/boundary; cell adhesion molecules stick cells together and to a particular tissue, often surrounded by a capsule so remain within tissues WHEREAS malignant tumours have irregular / poorly defined borders and not encapsulated; cells break off (+ grows projections into surrounding tissues) so metastasis occurs

- Benign tumours are easy to treat - can normally be removed by surgery, rarely returns WHEREAS malignant tumours have to be removed by radiotherapy / chemotherapy as well as surgery; can be life threatening and recurrence more likely

How do tumour suppressor genes normally function?

- Code for proteins involved in control of cell division

- In particular, stopping cell cycle (when DNA damage detected)

- Also involved in causing self-destruction of cell (apoptosis) (where damaged DNA cannot be repaired)

How can tumour suppressor genes play a role in the development of tumours?

• Mutation alters amino acid sequence and tertiary structure of protein = non-functional protein

- Increased methylation prevents transcription / expression of protein

- Damaged DNA not repaired / cells not killed; uncontrolled cell division

- Note - would need 2 mutated alleles

How do proto-oncogenes normally function?

- Code for proteins involved in control of cell division

- In particular, stimulating cell division (when growth factors attach to receptors on cell membrane, so cell division is required)

How do proto-oncogenes play a role in the development of tumours?

- Mutation could turn it into permanently activated oncogene

- Decreased methylation / increased acetylation causes excess transcription

- Cell division permanently activated; rapid / uncontrolled cell division

- Note - only need 1 mutated allele

How can increased oestrogen concentrations lead to the development of some breast cancers?

- Areas of high oestrogen conc. such as adipose tissues in breasts, cell division uncontrolled

- Growth of cancer minimised with drugs blocking production / action of oestrogens in the breasts e.g. Tamoxifen prevents oestrogen binding to receptor

What is the genome?

The complete set of genes in a cell

What are sequencing projects?

- Projects that have read the genomes of a range of organisms

- e.g. The Human Genome Project

Why are sequencing methods continuously updated and have become automated?

- Past - labour-intensive, expensive, could only be done on a small scale

- Now - automated, cost-effective and done on a large scale e.g. pyrosequencing

What are the applications of sequencing projects on simple organisms?

- Determining the genome of simpler organisms allows the assignment of proteins to each gene in the genome (proteome), creating a database

- Easy because less non-coding DNA

- Identifying the protein antigens on the surface of viruses / pathogenic bacteria can help in the development of vaccines

What are the applications of sequencing projects on complex organisms?

- Knowledge of the genome cannot easily be translated into the proteome, due to the presence of:

- Non-coding DNA

- Regulatory genes - determine when the genes that code for particular proteins should be switched on and off

- Human Genome Project - determined the sequence of bases of a human genome

What is recombinant DNA technology?

- The transfer of DNA fragments from one organism or species, to another

- Transferred DNA can be transcribed / translated into proteins within cells of the recipient (transgenic) organism, since the genetic code is universal and as are transcription and translation mechanisms

What are the 3 ways that fragments of DNA can be produced?

1. Conversion of mRNA to complementary DNA (cDNA), using reverse transcriptase

2. Using restriction enzymes to cut a fragment containing the desired gene from DNA

3. Creating the gene in a 'gene machine'

How are DNA fragments produced using the conversion of mRNA to complementary DNA (cDNA), using reverse transcriptase?

1. mRNA isolated from a cell that readily synthesises the protein coded for by the desired gene

2. Mix mRNA with DNA nucleotides and reverse transcriptase → reverse transcriptase uses mRNA as a template to synthesise a single strand of cDNA

3. DNA polymerase forms second strand of DNA (= double stranded = gene) using cDNA as a template

How are DNA fragments produced using restriction enzymes to cut a fragment containing the desired gene from DNA?

1. Different restriction endonucleases cut DNA at specific sequences of bases called a 'recognition sequence' - shape of recognition site complementary to active site

2. Some restriction enzymes cut in a staggered fashion = 'sticky ends' formed (uneven cut is left in which each strand of DNA has exposed, unpaired bases)

How are DNA fragments produced by creating the gene in a 'gene machine'?

- Synthesises fragments of DNA from scratch without the need for a pre-existing DNA template

- DNA fragments produced quickly / accurately

- Free of introns → can be transcribed by a prokaryote who can't remove introns

What are the advantages of using mRNA to make DNA fragment rather than restriction enzymes to cut gene from DNA?

- More mRNA in cell than DNA → easily extracted

- Introns removed by splicing (in eukaryotes) whereas DNA contains introns

- Bacteria can't remove introns

How can DNA fragments be amplified?

- In vivo methods

- In vitro methods

What are the 4 stages in the in vivo method of amplifying DNA fragments?

1. Promotor and terminator regions added to fragments of DNA

2. The use of restriction endonucleases and ligases to insert fragments of DNA into vectors

3. Transformation of host cells using these vectors

4. The use of marker genes to detect genetically modified (GM) cells / organisms

Why are promotor and terminator regions added to fragments of DNA?

- Promotor and terminator regions need to be added in order for the gene to be transcribed and then translated into a protein

- Promotor regions - DNA sequences that tell RNA polymerase when to start producing mRNA

- Terminator regions - tell RNA polymerase when to stop

Why are restriction endonucleases and ligases used to insert fragments of DNA into vectors?

- Vector transports DNA into host cell e.g. plasmids or bacteriophages

- Vector DNA and DNA fragment cut using same restriction enzyme

- Vector DNA and DNA fragment have complementary sticky ends → complementary base pair

- DNA ligase forms phosphodiester bond between adjacent nucleotides on sticky ends

What happens in the transformation of host cells using vectors?

- Host cells have to be persuaded to take in the plasmid vector and its DNA e.g. host cells placed into an ice-cold calcium chloride solution to make cell membranes more permeable, then plasmids added, and mixture is heat shocked

- Bacteriophage vector - infects host bacterium by injecting its DNA into it, then the phage DNA with target gene integrates into bacterial DNA

How are marker genes used to detect genetically modified (GM) cells / organisms?

- Not all cells / organisms will take up the vector and be transformed

- Marker genes, inserted into vectors at same time as target gene, are added in order to identify which cells have the desired gene

- Gene markers can be:

- Resistance to an antibiotic

- Fluorescent protein

- Enzyme whose action can be identified

How does the PCR work as an in vitro method?

Amplification specific DNA fragments (in every cycle, amount of DNA doubles)

What is the reaction mixture in the PCR?

- DNA fragment

- DNA polymerase (taq polymerase)

- (forward/reverse) Primers

- Nucleotides

How many times is the cycle repeated in the PCR?

30-40 times to make lots of copies of DNA fragment

What are the 3 stages of the PCR?

1. Strand separation (95C)

2. Primer annealing (55C)

3. DNA strand synthesis (72C)

What happens in strand separation (95C)?

What happens in primer annealing (55C)?

- Allows primers to bind / anneal to DNA fragment template strand, forming hydrogen bonds

- Primer = short, single stranded DNA fragment

- Primer complementary to template DNA at edges of region to be copied

- DNA polymerase binds to primer to start synthesis; it can only add nucleotides onto pre-existing 3' end

- Two different primers required (forward and reverse)

- Because DNA strands run in anti-parallel, but polymerase can only run in one direction

What happens in DNA strand synthesis (72C)?

- Optimum temperature for DNA polymerase to make complementary copies of DNA

- Nucleotides align next to complementary exposed bases

- DNA polymerase joins adjacent nucleotides, forming phosphodiester bonds

What are DNA probes and how do they work?

- Short, single stranded pieces of DNA that are labelled radioactively or fluorescently so they can be identified.

- This is used to locate specific alleles of genes and to screen patients for heritable conditions, drug responses or health risks.

- DNA probes are created to have a complementary base sequence to the allele that is being screened for.

- The patients DNA sample is treated to make it single-stranded and it is then mixed with the DNA probes.

- If the patient has the allele, then the DNA probe will hybridise and the label indicates the presence

What happens in DNA hybridisation?

- The patients DNA sample is heated to make it single stranded.

- The hear causes the hydrogen bonds between bases to break (denaturing)

- The patients single stranded DNA sample is mixed with the DNA probe, and cooled and any complementary sequences can align and form hydrogen bonds (anneal)

- Some of the patients DNA samples will anneal back together, but some will anneal with the DNA probe.

How can specific alleles of genes be located?

1. To locate a specific allele, the DNA base sequence must be known to then create a DNA probe.

2. This can be determined using DNA sequencing techniques.

3. The fragment of DNA can be produced using a gene machine.

4. This fragment can be amplified using PCR

5. The label is then added, either a radioactive nucleotide containing the isotope 32P, or a fluorescent label which emits light under UV light.

6. After hybridisation, the DNA is washed so that any unbound DNA probes are washed away.

7. The presences of the radioactive or fluorescence label therefore indicates that the allele of interest is present in the patients DNA.

What is genetic screening?

- When DNA probes are used to screen for potential genetic disorders of for the presence of cancer causing oncogenes.

- It is possible to screen for multiple diseases simultaneously using an array, where multiple different DNA probes are attached.

What uses can DNA screening have?

- Personalised medicine - certain drugs such as painkillers are more or less effective depending on your genotype It can also help determine the best dose, which increases the effectiveness, safety and save money.

- Genetic counselling - patients are informed of any potential risks to themselves or future offspring if they were to carry an allele linked to diseases. Before starting a family or for general health.

What are variable number tandem repeats?

- Regions found in the non-coding part of DNA

- A short DNA sequence (e.g. GATA) is repeated a variable number of times without any spaces, at a single location

- The probability of two individuals having the same VNTRs is very low, however the more closely related you are the more similar the VNTRs are.

What is genetic fingerprinting?

The analysis of VNTR DNA fragments and this can be used to determine genetic relationships and the genetic variability within a population.

Describe how genetic fingerprinting is carried out

1. DNA extracted from sample;

2. DNA cut / hydrolysed into segments using restriction endonucleases;

3. must leave VNTRs intact;

4. DNA fragments separated using electrophoresis;

5. mixture put into wells on gel and electric current passed through;

6. immerse gel in alkaline solution / two strands of DNA separated;

7. Southern blotting / cover with nylon / absorbent paper (to absorb DNA);

8. DNA fixed to nylon / membrane using uv light radioactive marker / probe added (which is picked up by required fragments) / complementary to VNTRs;

What are the uses of genetic fingerprinting?

- Forensic science to place suspects are crime scenes

- For medical diagnosis

- To ensure animal and plants are not closely related before being bred

What is the difference between DNA polymerase and DNA ligase?

- DNA polymerase joins INDIVIDUAL DNA nucleotides together, working along a template strand in DNA replication.

- DNA ligase joins DNA FRAGMENTS together at a single point im genetic engineering