Biology 3.8
3.8.1 Mutation
2 types
Chromosome mutation is a change in either the number of chromosomes or a change in the overall structure of a chromosome
Gene mutation is a change in the sequence of bases on the DNA
Causes
Spontaneous mutations caused by random mistakes during DNA replication, occur naturally with a low frequency
Rate increased by exposure to mutagenic agents
Eg. X-rays, UV light, she chemicals (benzpyrene in tobacco)
Types
Frameshift
Addition
One or more nucleotide bases added into sequence
Deletion
One or more nucleotide bases is deleted from the sequece
Non-frameshift
Substitution
One nucleotide base is exchanged for another
Only onetriplet codon is affected
Consequences
Nonsense mutations
Codon is changed into a stop codon so translation is stopped prematurely, so protein is only partly formed + non-functional
Mis-sense mutations
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
Silent mutations
Have no effect
Mutation in non-coding intro or mini-satellite
Genetic code is degenerate so same amino acid produced
Examples
Cystic fibrosis
Sickle Cell Anaemia
Phenylketonuria
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
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
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
3.8.2.1 Potency + Stem Cells
General
Cells of a new individual become specialised by differentiation
Only some of the genes are expressed
Only part of the DNA is expressed + translated
Definitions
Totipotent
Cells which retain the ability to form all parts of a mature organism Eg. Zygote
Plants
In many species differentiation is reversible
In Vitro Cloning or Micropropagation
Micropropagation
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
Animals
Specialisation is irreversible
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
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
Stem cells
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
3.8.2.2 Regulation of Translation + Transcription
Operon system
In many prokaryots gene expression is controlled by special switches Eg. Utilisation of lactose in E. Coli
Involves 3 genes:
-Structural: Encodes 3 enzymes for uptake+breakdown of lactose
-Operator: Located before+switches on structural, contains promoter sequence for RNA polymerase to bind
(Operon=Structural+Operator)
-Regulator: Codes for repressed molecule
When bacterium grown on glucose medium, regulator gene produces repressor protein - binds to operator gene+switches off=structural genes not switched on+enzymes not produced
When bacterium on lactose medium, lactose combines with repressor protein=operator+structural genes active+enzymes produced
Transcriptional factors
Definition: A regulatory protein that moves from the cytoplasm into the nucleus+binds to DNA at a specific point
Stimulates transcription of specific genes, producing mRNA which can be translated into a protein
If there is no ranscriptional factor, gene is effectively switched off as transcription cannot begin
Eg. Effect of oestrogen
Oestrogen produced in ovaries+carried in blood stream
Binds to specific complementary receptors on csm
Lipid soluble so can diffuse across phospholipid bilayer
Binds to receptor molecule in transcriptional factor
Transcriptional factor changes shape+inhibitor falls off
TF moves into nucleus through nuclear pore
Binds to DNA+stimulates transcription of that area
Eg. Haemoglobin composition varied due to different genes being expressed at different times with different transcriptional factors
Affects oxygen affinity of the molecule
Epigenetics
Definiton
"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
Epigenome
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
RNA interference
Gene expression can be regulated after transcription by breaking down mRNA before it can be translated
Small interfering RNA (siRNA) molecules consist of pieces of double stranded RNA
Long double sided molecules of RNA are transcribed
Dicer enzyme cuts RNA into short sections
One stands broken down, the other is the guide strand
Guide combines with enzyme complex to form RISC complex
Guide leads RISC to bind to specific mRNA molecule+H bond
mRNA broken down by slicer enzyme in RISC
Can no longer be translated so gene is effectively silenced
May also be important in immune response to viral infection
Could have therapeutic value
3.8.2.3 Cancer
Differences between benign+malignant tumours p519
Cells begin to divide in an uncontrolled way, forming tumours
Tumours are unable to carry out normal functions of a cell so functioning of tissues affected
Tumours may spread into neighbouring tissues+block blood vessels/nerves+disrupt organized function
Tumour cells may break off+be transported to other sites where they develop into secondary tumours (Metastases)
Changes that result in cancer can be epigenetic changes or as a result of mutations in key genes
Carcinogens
Cancer-causing agents with damage DNA causing changes in the base sequence
Examples:
Ionizing radiations, Chemicals, Viruses, Cells own metabolic products, inherited predisposition
Cancer does not develop more often because:
More than 1 mutation is required for a cell to become cancerous
Control genes are small fraction of DNA=low chance of being hit
Efficient surveillance+repair system recognises damaged DNA
Apoptosis=extensively damaged cells destroyed by immune sys
Cell cycle
Tightly controlled by:
Proto-oncogenes-code for proteins which simulate cell divison
Tumour suppressor genes-code for proteins which inhibit cell division, repair DNA+trigger apoptosis
Problems
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
3.8.3 Genome Project
Definitions
Genome: All of the genetic material in an organism
Proteome: All of the proteins produced from the genome in a given type of cell at a given time under specific conditions
Genome projects are designed to determine the base sequence of the DNA
DNA sequencing
Sanger sequencing technique/Chain termination technique/Dideoxy technique
Makes use of the fact that if an abnormal nucleotide is placed onto a growing Chan during replication, replication will stop
Method:
-4 reaction tubes contagious unknown DNA, radioactivity labelled primers, normal nucleotides, abnormal terminator nucleotides (each has a differentterminaor nucleotide)
-DNA polymerase added+DNA begins to replicate
-Replication begins then stops at abnormal terminator
-Each strand will terminate in a different place so fragments a different lengths
-Fragments loaded onto gel +separated using electrophoresis
-Smaller fragments move furthest
-Photographic film is laid on top to visualize bands (dark bands)
-Film is read from the bottom upwards
Whole-genome shotgun method is used to determine sequence of the entire genome of a organism
-Cut genome into short sequences
-Sequence fragments using Sanger Technique
-Computer programmes align overlapping fragments
It is relatively simple to determine the genome of prokaryots as it consists of a single chromosome + no non-coding sequences
Eukaryot DNA contains non-coding intros + mini-satellites + some genes have a regulartory role, don't encode for functional proteins
3.8.4.1 Recombinant DNA Technology
In Vivo Gene Cloning
In vivo gene cloning/genetic engineering/recombinant gene technology = All of the techniques involved in extracting + transferring a gene from donor organism to recipient/transgenic organism
Used for:
Transferring genes so an organism will produce a useful gene product
Inserting genes to improve features of an organism
Studying how genes work
Method
Isolation
Ligation
Transformation
Selection
Culturing
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
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
-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)
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
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
Genetic modification
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
In Vitro Gene Cloning
Polymerase Chain Reaction
-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
Gene Therapy
Involves treating genetic disorders by inserting a copy of a normal gene into a target cell in order to restore cell function
Germ Cell Therapy
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
Somatic Cell Therapy
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
Evaluation
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
3.8.4.2 Identifying + Diagnosing Inherited Conditions
3.8.4.3 Genetic Fingerprinting
Useful diagnostic tool in forensic science + determining relationships
95% human DNA is non-coding
Mini-satellites between genes
Intros within genes
Contain hypervariable regions which consist of short base sequences (core nucleotide sequences) which repeat
Number + length of repeats is highly individual
More related = more similar sequences
Method
Extraction
Tissue damage mixed with solvent (chloroform or water saturated phenol) which dissolves DNA + precipitates out protein
PCR used to amplify sample
Digestion
DNA cut using specific restriction endonucleases
Cut close to hypervariable regions
Releases fragments of varying sizes
Separation
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
Hybridisation + development
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
Finding genes
DNA Probes + Hybridisation
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
DNA Microarrays
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
Genetic Testing
Why
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
Allows
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
How
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