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Biology 3.8 (3.8.2.3 Cancer (Carcinogens (Cancer-causing agents with…

- - - - Proto-oncogenes
        
        Normally:
        -Code for a complementary receptor protein in the cell membrane which allows a growth factor to bind+trigger cascade of proteins which leads to genes for DNA replication being switched on
        -May code for growth factors themselves
        -May code for relay proteins
        
        Mutated:
        Called Oncogene
        -Change in receptor protein so it activates sequence without growth factor present
        -Excess growth factor produced=over activation of cell division
        -Change in any relay protein so they are permanently activated
      - Tumour suppressor genes
        
        Normally:
        Proto-oncogenes are switched off because they are inhibited by Tumour suppressor genes
        
        Mutated:
        No longer functions are proto-oncogenes a not inhibited+are expressed continually=uncontrolled cell division
      - DNA repair genes
        
        Some genes code for enzymes which repair damaged DNA
        
        If these become damagedthemselves, repair system will not function+mutations will accumulate
      - DNA methylation
        
        Hypermethylation of tumour suppressor genes
        Chromatin is more tightly packed so genes are inaccessible
        Gene is inactive=uncontrolled cell division
        
        Hypomethylation of proto-oncogenes
        Chromatin is loosely packed so genes available for transcription
        Gene active=uncontrolled cell division
- - - - Addition
        
        One or more nucleotide bases added into sequence
      - Deletion
        
        One or more nucleotide bases is deleted from the sequece
    - - Substitution
        
        One nucleotide base is exchanged for another
        Only onetriplet codon is affected
  - - - Codon is changed into a stop codon so translation is stopped prematurely, so protein is only partly formed + non-functional
    - - Altered mRNA is produced during transcription then translated into an altered sequence of amino acids to form an altered protein (altered tertiary structure)
        Benefical: Results in an advantageous protein
        Harmful: Produces non-functional protein
    - - Have no effect
        Mutation in non-coding intro or mini-satellite
        Genetic code is degenerate so same amino acid produced
    - - Cystic fibrosis
        
        Genetic disease caused by a recessive mutation in agent which codes for a channel protein called the Cystic Fibrosis Transmembrane Regulator in epithelial cells
        
        Mutation is a deletion of 3 bases meaning that one of the amino acids normally present in the CFTR protein is missing
      - Sickle Cell Anaemia
        
        Single-point mutation
        A 'Val' amino acid (non-polar) replaces a 'Glu' amino acid
        Decreasing the solubility of a haemoglobin molecule
        Red blood cells become a sickle shape = cannot carry oxygen efficiently + get stuck in capillaries
        Areas of tissue become deprived of oxygen + cells die
      - Phenylketonuria
        
        Inherited disease caused by a mutation in the gene coding for phenylalanine hydroxylase
        
        Phenylalanine will accumulate + cause brain damage in children
        Insufficient melanin forms so sufferers are very light
- - - - "The study of potentially heritable changes in gene expression the does not involve changes to the underlying DNA base sequence"
        A change in phenotype without a change in genotype
      - Some phenotypic changes which he caused by environmental factors are inherited even though they do not actually change the base sequence on the DNA
    - - Eukaryotic DNA is wrapped around hisone proteins forming Chromatin
        They are associated with epigenetic tags (Chemical groups)
        Epigenome shapes the physical structure of underlying genome
        Where chromatin is very tightly wrapped, genes are inaccessible
        Genes a switched off (unreadable)
        Where chromatin is relaxed, genes are accessible to TF (on)
        Structure of the Epigenome can change in response to environmental factors, whereas structure of DNA is fixed
      - A cell's epigenetic profile is the up of the chemical signals the cell receives during its lifetime, from inside the cell, neighbouring cells or the environment (Not all tags are inherited-cell reprogramming)
      - Once a signal reaches a cell+attaches to a specific receptor, this triggers a system of relay proteins which effectively transmit the information to the nucleus of the cell, then ultimately to a gene regulatory protein the attaches to a specific DNA sequence
      - Regulatory protein:
        
        Switches specific genes on or off
        
        Protein attaches to a specific DNA sequence+acts as a switch
        
        Recruit enzymes that add+remove epigenetic tags
        
        Acetyl groups
        
        Addition adds -ve charge to histone proteins
        Strongly repelled by phosphate groups on DNA
        Loose chromatin=Genes turned on
        
        Removal adds +ve charge to histone proteins
        Strongly attracted to phosphate groups of DNA
        Tightly packed chromatin=Genes turned off
        
        Methyl groups
        
        Added to Cytosine base
        Increase association between DNA+histones
        Tightly packed chromatin=Genes turned off
        
        Demthylation
        Decreases association of DNA+histones
        Loose chromatin=Genes turned on
- - - - Totipotent
        
        Cells which retain the ability to form all parts of a mature organism Eg. Zygote
      - Pluripotent
        
        Cells have the ability to form every cell in the adult mammal except the placenta
      - Multipotent
        
        Cells can differentiate into a limited number of cells
      - Unipotent
        
        Cells can only differentiate into a single type of specialised cell
        Produced from multipotent cells + founding adult tissues
  - - - Method
        
        Donor plant tissue (Explant) is taken from parent plant
        Surface is sterilised in sodium hypochlorite then rinsed
        Transferred into a sterile container with growth medium
        (sucrose, amino acids, vitamins, inorganic nutrients, cytokinins)
        Shoots subdivided to produce more new shoots
        Transferred into medium containing growth hormones: auxins
        Platelets with roots translated into sterile compost
        Temperature + Light intensity are appropriately controlled
      - Advantages
        
        Cloned plants are genetically identical to parent
        Many copies with desirable characteristics produced quickly
        Some plants have low efficiency reproduction
        Some plats are difficult to grow from seeds
        Rapid production from a few stock plants
        Plants can be grown at any time of year
      - Disadvantages
        
        Labour-intensive processes = high labour costs
        Expensive equipment = high set-up cost + expensive product
        Requires trained staff
        Unforeseen problems may arise as techniques are relatively new
  - - - Zygote is totipotent
        After about 5 days cells begin to differentiate
        Form a blastocyst (hollow ball of cells)
        -Outer layer (trophoblast/placental stem cells) will form placenta
        -Inner layer (embryonic stem cells) are pluripotent
        Pluripotent cells divide to 2 daughter cells
        
        1 forms a differentiated cell
        
        1 forms a multipotent adult stem cell
    - - Key features
        
        Self-renewal: Ability to divide + remain undifferentiated
        Can be maintained in a lab culture
        
        Degree of potency: Can differentiate into a range of specialised cell types
      - Embryonic stem cells
        
        Issues
        
        Rights of the embryo
        Who gives consent
        
        Source of the embryo
        
        Unused embryos from IVF
        
        Embryos made from donated eggs
        
        Embryos produced via therapeutic cloning
        
        Could create tissue derived from the patient's own cells so would not cause rejection
        
        Genetic differences could trigger rejection
        
        Cells from so early stage embryo are cultured in vitro
        Can develop into different tissues when given special differentiation factors
      - Adult stem cells
        
        Disadvantages
        
        Only multipotent so can only make a limited range of cells
        More limited self-renewal = less stable
        Can be difficult to separate cells from original tissue
        
        May have some therapeutic value
        
        Advantages
        
        No embryos are destroyed
        No rejection
      - Induced pluripotent stem cells (iPS's)
        
        Some cells can be artificially induced to become pluripotent stem cells
        Given the correct growth conditons, iPS cells could differentiate into a wide variety of specialised cells
        
        Uses
        
        Disease modelling
        
        Drug effectiveness evaluation + toxicity testing
        
        Cell therapy
        Patient's own cells treated to become iPS cells then stimulated to differentiate into specific cells to treat a range of conditions
- - - - Isolation
        
        Restriction Endonucleases
        
        -Cell membrane broken down with detergent
        -Centrifuged - DNA+associated proteins collected
        -Proteins digested by proteases to leave DNA
        -DNA cut into fragments by restriction endonucleases
        -Fragment carrying desired gene needs to be identified
        -Separate fragments on size+identify with radioactive gene probe
        
        Enzymes which occur naturally in bacterial cells
        Cut at specific target recognition palindromic sequences
        Some cut straight across to leave frames with blunt ends
        Some cut asymmetrically leaving sticky ends
        Can be stuck into vector with complementary sticky ends
        
        Problem: Prokaryot cells cannot splice so if the entire gene is added, it will all be transcribed + resulting message is useless
        
        Reverse Transcriptase
        
        Isolate mature mRNA produced from desired gene
        
        Eg. Insulin
        -Mature mRNA isolated from pancreas cells
        -Mixed with reverse transcriptase which makes a DNA copy from the RNA template (opposite of transcription)
        -Hybrid molecule of mRNA + DNA produced
        -Alkali destroys mRNA to leave single strand copy DNA (cDNA)
        -DNA polymerase converts single strand to double strand
        -Promoter sequence added to beginning of gene
        
        Artificially Synthesising
        
        Artificially synthesise a gene for genetic engineering
        Use machine that synthesises DNA from nucleotides
        Depends on knowing the base sequence of the gene
        If order of amino acids is known, bases can be worked out
      - Ligation
        
        Inserting the gene into a vector = bacterial plasmid
        
        Vector = carrier DNA molecule into which desired gene can be inserted to produce a recombinant molecule
        
        Plasmid = Small circular piece of double stranded DNA which occurs naturally in many bacteria, separate from the bacterial chromosome + often carry genes for antibiotic resistance
        Most common type of vector
        Replicate independently
        Antibiotic resistance genes can be useful markers
        
        -Plasmid cut open using same restriction endonucleases to give same sticky ends sequence
        -Plasmids + gene fragments mixed together + H-bonds form
        -DNA ligase seals gaps in sugar-phosphate backbone
      - Transformation
        
        -Recipient bacteria put in cold calcium chloride for 30mins to make cell membrane+wall more permeable to DNA
        -Bacteria mixed with recombinant plasmids + warmed to stimulate bacteria to take up plasmids (low efficiency)
        -Identify bacteria containing recombinant plasmid using antibiotic resistance genes as markers
        (Inserting a new gene will inactivate a resistance gene)
      - Selection
        
        Replica plating
        -Sample of culture placed on agar gel containing ampicillin
        -Only bacteria containing a plaid will grow
        -This master plate is replica plated
        -Sterile disc lowered onto surface so some colonies will stick
        -Disc lowered onto agar plate containing tetracycline
        -Bacteria with recombinant plasmid will not grow
        -Compare plates to find bacteria with recombinant plasmid
        
        Markers:
        Antibiotic Resistance: New gene disrupts resistance gene
        Fluorescent Markers: New gene disrupts GFP gene
        Enzyme Makers: New gene disrupts lactase enzyme gene
      - Culturing
        
        Desired bacteria cultured in an industrial fermenter
        -Sterile equipment = only desired bacteria
        -Culture medium provides nutrients (carbs, protein, vits+mins)
        -Mixed = good mixing of bacteria + nutrients
        -Well aerated with sterile air = oxygen for aerobic respiration
        -Temperature + pH monitored +maintained at optimum
        -Cooling jacket = prevent overheating (bi-products of respiration)
        
        Desired gene will be transcribed with other genes during protein synthesis + gene productbis manufactured in large quantities then can be isolated
    - - Modifying the original genome of an organism to improve its characteristics, developing transgenic organisms
        Move genes using: liposomes, Plasmid vectors, viral vectors, ballistic DNA injection, protoplast fusion
      - Plants
        
        Eg. Flavr Savr Tomato, Plants that repel insects, Herbicide tolerant
        
        Against:
        -Toxins may build up in food chains with unknown consequences
        -Toxins may be released into soil during decomposition
        -Useful insects may also be killed
        -Herbicie resistance may be passed on
        -Human side effects are unknown (allergic reactions)
        -Antibiotic resistance genes may be transferred to bacteria
        
        For:
        -Speeds up selective breeding
        -Precise + only transfer select number of genes
        -Increased yields, less wastage + better quality = more fed
        -Develop crops able to grow in poor quality soil
        -Reduce use of insecticides
      - Animals
        
        Modified to improve characteristics or produce gene product
  - - - -DNA heated to 95C to break H bonds + form separate strands
        -Primers (short DNA/RNA sequence=starting sequence) added
        -Cooled to 55C so primers bind
        -Thermostable DNA polymerase enzyme from hot spring bacteria added with some nucleotides
        -Heated to 72C = enzyme binds to primers + copies strands
        -Mixture heated again to separate new DNA + cycle repeated
      - Used to amplify samples of DNA
        Genetic fingerprinting in forensic science
        Genetic fingerprinting in paternity disputes
        Genetic engineering procedures
        From fossils+dead remains for comparisons
  - - - Therapy of egg, sperm or early embryo
      - Problem: Results in altering genotype of every cell inc subsequent gametes = alteration is passed on to next generation
        Since long them effects of gene therapy treatments are unknown, gem cell approach is currently unacceptable
    - - Therapy of body cells other than germ cells
        Mostly involves gene supplementation = delivering a Norma copy of a gene to target cell without removing defective gene
      - In Vivo = Desired gene introduced into vector which carries it to target tissue in patient's body
        Eg.Treatment of cystic fibrosis (non-viral liposome+adenoviruses)
        Ex Vivo = Desired gene inserted into vector which inserts it into target cells removed from patient, then returned to patient
        Eg.Treatment of SCIDS (retrovirus vector)
      - Vector systems
        
        Viral vectors
        
        Viruses consist of nucleic acid surrounded by protein coat designed specifically for entering cells + expressing genes
        "Tamed" viruses can transfer genes without causing disease
        
        Retro viruses
        
        Capable of inserting genes into host cell chromosomes so offer long term stability
        Target a wide range of cells
        Large virus can carry large gene
        
        Problems:
        Promiscuous= Infect variety of cells+targeting is difficult
        Insert genes randomly so could disrupt important host genes
        Body may develop immune response to virus = kill host cells
        
        Adenoviruses
        
        Very efficient infecting human cells+don't cause serious disease
        Large viruses can carry large genes
        
        Problems:
        Do not splice genes into chromosomes but deliver to nucleus
        =genes effective temporarily
        Lack of specificity in infection
        Strong immune response kills altered cells = treatment may fail
        
        Adeno-Associated viruses
        
        Cause no known disease + no immune response
        Integrate genes into host cell chromosomes
        
        Problem:
        Small so can only carry very small genes
        
        Problems:
        Target specificity: Infect a variety of cells but can overcome this by modifying viral envelope to contain receptors specifically recognises by desired target cells
        Tamed virus could alter inside body + become harmful
        Virus may disrupt host cell genes with potentially harmful effects
        Immune response may kill modified cells
        
        Non-viral vectors
        
        Liposomes
        
        Therapeutic gene is spliced into bacterial plasmid which carries it into host cell nucleus
        Plasmid is coated in non-immunogenic lipid which is +ve charged
        Lipid fuses with target cell membrane + carry plasmid into cell
        
        Problems:
        Getting lipid to associate with plasmid
        Lower target cell infection efficiency
        
        Advantages:
        Do not cause disease
        Do not disrupt host cell gene function
        
        Polyplexes
        
        Use an amino acid polymer to encapsulate the plasmid
        So far only been used in tissue culture
        
        Naked DNA
        
        Can be taken up by + expressed in muscle cells
        Potential for gene therapy vaccines against HIV, herpes, malaria
    - - Successful treatment of difficult disorders+diseases
      - Problems:
        Finding+maintaining stable vectors which will not interfere with normal host cell get function, mutate backstop cause disease or be immunogenic
        Targeting vectors to specific cells
      - Ethical issues:
        Germ cell therapy
        Lead to human genetic modification
        Could funds be better spent
- - - - Tissue damage mixed with solvent (chloroform or water saturated phenol) which dissolves DNA + precipitates out protein
        PCR used to amplify sample
    - - DNA cut using specific restriction endonucleases
        Cut close to hypervariable regions
        Releases fragments of varying sizes
    - - Gel electrophoresis separates based on size
        Mixture loaded into wells in agarose gel
        Placed in electrophoresis tank in pH7 buffer solution
        Voltage applied across gel
        -ve phosphate groups in DNA attracted to +ve gel end
        Smaller fragments move quicker
        Fragments of known size used for calibration
        Gel immersed in alkali to form single stranded DNA
        Fragments transferred to thin nylon membrane by Southern Blotting=thin nylon membrane placed on gel, blottingbpaer place on top, buffer drawn up by capillary action, bringing DNA fragments, which stick to membrane in same position as on gel
        DNA fixed to nylon by exposure to UV light
    - - Radioactive DNA probes used to visualise DNA position
        (Short base sequences complementary to desired sequence)
        Nylon membrane incubated with probes
        H-bond to fragments with desired sequence
        Nylon rinsed to remove unbound fragments
        Covered with photographic film: dark line = radioactivity
      - Alternative method:
        Probes labellednwith alkaline phosphatase enzyme
        After hybridisation, membrane covered with phosphate-containing substrate
        Enzyme removes phosphate group causing it to fluoresce
        Position of bands seen
- - - - DNA probe = single strand of DNA that he complementary base sequence to the known sequence being detected
        (At least part of the mutated sequence must be known so correct probe can be synthesised)
      - Labelled with:
        Radioactive nucleotides containing 32P
        -Can be detected using photographic film
        Fluorescent label
        -Can be detected under UV light
      - Method:
        Specific complementary labelled probe manufactured
        Test material collected
        DNA isolated + treated with restriction endonucleases to make fragments + treated to make single stranded
        Fragments separated using gel electrophoresis
        Transferred to nitrocellulose sheet by Southern blotting
        Sheet placed in container + probe added
        Probe can H Bond if mutated sequence present
        Unbound probe washed off
        Probe detected by presence of label
    - - Use DNA probe technology
        Can quickly compare known DNA which is bound to microarray to unknown DNA
      - Method:
        mRNA collected from cells
        Reverse transcriptase makes cDNA copies
        Labelled with fluorescent dye
        Added to microarray
        cDNA H-bonds to specific probe so spot will fluoresce
        Computer analysis determines which genes are on/off
  - - - Find outbid they are carrying a mutation which may be expressed at a later date
      - Find out if they are an asymptomatic carrier of a recessive mutation
      - Find out if they are carrying a recessive mutation which may make them predisposed to developing a particular condition
    - - Lifestyle alterations to avoid predisposed conditions
      - Regular checks for conditions -> earlier diagnosis + treatment
      - Choose most appropriate form of treatment
        Use gene therapy treatment if appropriate
      - Make informed decision about reproduction
    - - Pre-natal screening
        
        Amniocentesis
        
        Fine hyperdermic needle collects sample of amniotic fluid with some foetal cells which can be tested
        13-16 weeks
        
        Chorionic villus sampling
        
        In catheter takes sample from actively dividing cells of the chorionic villi of the placenta
        9-12 weeks
      - Pre-Implantation Genetic Testing
        
        Uses IVF techniques tonproduce embryos in the lab
        Cells removed from embryo + screened