Module 6: Genetic Change
• Mutation: permanent change to genome (i.e. DNA nucleotide sequence)
• Mutagen: environmental agents that alter DNA
o Mutagenesis under prolonged exposure → induced mutations
o Chance of mutation increases with frequency & length of exposure
Explain how a range of mutagens operate, including:
Physical mutagens
Electromagnetic radiation
• Energy transfer from Sun
• ↓ wavelength = ↑ energy
o ↑ ionisation (removes electrons)
• UVB & UVC radiation (180-315 nm)
o Cross-linked nucleotides → forms pyrimidine dimers (C, T)
▪ Can’t pair with complementary base
▪ Strand ends prematurely
o Releases free radicals
▪ Double-strand DNA breakages → deletion of genetic material
▪ Oxidative stress
Other sources of ionising radiation
• Radiation from atomic bombs, medicine (X-rays, gamma rays) etc.
• Cosmic rays from Sun & outer space
Chemicals
• Alcohol, tobacco smoke, industrial solvents (benzene)
Base analogues
• Similar structure to nitrogenous base
• Mispairing during replication (inserts wrong complementary base)
Alkylating agents (eg. sulfur mustard)
• Adds alkyl groups (eg. -CH3) to nucleotides
• Strand terminates prematurely
Intercalating agents (eg. proflavine)
• Inserted into sequence → frameshift mutation
• Results in missense mutation (codes for incorrect amino acid)
o Dysfunctional protein
Lili Wang (2019)
Naturally occurring mutagens
• Present in normal levels within natural environment
Biological mutagens
• Microbes (eg. HIV, Helicobacter pylori)
o Insert genome into host cell
▪ Inflammation → releases free radicals
▪ Oxidative stress
o Disrupt host DNA + repair mechanisms
• End-products of metabolism
o Nitrosamine (carcinogenic)
▪ Reaction between nitrosating agents & amines (eg. cured meats)
▪ Activated by direct heat
▪ Linked to colorectal cancer
Non-biological mutagens
• Metals (eg. mercury)
Compare the causes, processes & effects of different types of mutation, including:
Causes of mutation
Mutagen-induced • Prolonged exposure to mutagenic agents (eg. UV radiation)
Spontaneous Non-disjunction
(eg. replication error in • Homologues or sister chromatids don’t separate properly
S phase) • Gametes with abnormal no. of chromosomes
o Affects 2 or 4 (anaphase I/II)
• During early embryonic development = mosaic syndromes
• Klinefelter syndrome (XXY)
o Abnormally small testes + sterile
Unequal crossing over
• Repetitive DNA → unaligned non-sister chromatids in tetrad
o Duplication/deletion in either gamete
Transposons (40% of genome)
• ‘Jump’ to different loci within genome
o ‘Cut & paste’ or ‘copy-paste’ transposition
• Prone to unaligned homologous pairing in meiosis
o Surrounded by inverted repeats
o Duplicated DNA in gamete
• Damages functional gene if inserted
o L1 retrotransposon inserted into factor VIII gene = haemophilia A
Lili Wang (2019)
Mutations in regulatory/repair genes
• Mutated proto-oncogenes
o Point mutation in promoter region, activates oncogenes
o Increases cell division = cancerous tumours
• Mutated tumour suppressor genes (eg. BRCA1)
o Mutation causes underactivity (tumours) or overactivity (atrophy)
• Mutated DNA repair genes
o Causes replication of mutated DNA → cancer
Point mutation
• 1 nucleotide varies by 1 base in 1 gene
Processes
• Base-pair substitution: replaces complementary nucleotides with another pair
• Insertion/deletion of nucleotides: shifts RNA ‘reading frame’
o If not occurring in multiples of 3, leads to frameshift mutation
• Inversion: nucleotide pair reverses position with another pair
Effects
• Missense mutation: produces different AA
o Refer to sickle cell anaemia
• Nonsense mutation: changes AA into stop codon
o Early termination of polypeptide
o Non-functional protein (eg. enzyme with incorrect active site)
• Silent mutation: no AA change
o Redundancy in genetic code
o No observable effect on phenotype
Example
• Sickle-cell anaemia
o Substitution mutation of A for T in beta-globin gene (chromosome 11)
▪ 6th AA = GAG → GTG
▪ Codes valine, not glutamic acid
o Distort haemoglobin into rigid rods = deoxygenated
▪ Periodic episodes of pain
▪ Painful swelling of hands etc.
o However, survival advantage against malaria
Lili Wang (2019)
Chromosomal mutations
• Permanent change to chromosomal structure or number
o Rearranges, deletes or disrupts loci
Processes
• Deletion: removes DNA section = less genes
o Deletion of chromosome 5’s short arm → cri du chat syndrome
• Duplication: replicates DNA section = more genes
o Phenotypic effect depends on size, location & no. of repeats
▪ Excess trinucleotide repeats = trinucleotide disorders
▪ Could become unaligned at crossing over
• Lead to deletions/insertions
• Inversion
o DNA section breaks off & reattaches in reverse orientation
▪ Inversion of X chromosome’s factor VIII gene → haemophilia A
• Translocation
o DNA section transferred to non-homologous chromosome OR new site on same chromosome
Effects
• Aneuploidy
o Abnormal no. of chromosomes in each cell
o Fertilisation of abnormal & normal gamete
▪ Monosomy: (2n-1)
▪ Trisomy: (2n+1)
• Polyploidy
o >2 complete sets of homologous chromosomes per nucleus
o Triploid = 3 sets
Example
• Huntington’s disease
o Autosomal dominant
▪ >40 trinucleotide CAG repeats in HTT gene (chromosome 4)
▪ Neurological abnormalities (eg. involuntary jerking, irritability)
Lili Wang (2019)
Distinguish between somatic mutations & germ-line mutations & their effect on an organism.
Somatic mutations
• Within somatic cells (non-reproductive)
o Not inherited by descendants
o Leads to atrophy (tissue dieback) or cancer
• Mutations in non-coding regions affect gene regulation (link to M5)
Germ-line mutations
• Occurs in germ cells (which undergo meiosis to form gametes)
o If mutated gamete is fertilised into zygote, mutation is inherited by offspring
▪ 100% mutated cells (eg. all cells with 3 copies of chromosome 21)
Example
• Phenylketonuria
o Mutations in gene that codes for phenylalanine hydroxylase (chromosome 12)
▪ Enzyme that breaks down phenylalanine (Phe) into tyrosine (Tyr)
o Causes toxic accumulation of phenylpyruvate
▪ Mental retardation, seizures, spasms
• Down syndrome
o Mostly not inherited
▪ Non-disjunction of chromosome 21 (92%)
• Gamete with 2 copies → fertilised into zygote (3 copies)
• If occurring in early embryogenesis, leads to mosaic DS (some normal cells)
▪ Translocation of chromosome 21 to 14
o Physiological & neurological abnormalities (eg. heart defects, learning disabilities)
Assess the significance of ‘coding’ & ‘non-coding’ DNA segments in the process of mutation.
Mutations in coding DNA
o Affects AA sequence (eg. nonsense, missense)
o ‘Conservative’ mutation = substitutes for biochemically similar AA
o ‘Non-conservative’ = deleterious
o In recessive gene: not expressed in heterozygotes
Coding DNA is transcribed into protein sequences (i.e. polypeptides).
Mutations in non-coding DNA
o Do not code for AA sequences
o Affects gene expression
o Regulatory sequences (eg. promoters: binding sites for TF)
o Transcribed into RNA molecules (eg. tRNA)
Lili Wang (2019)
Effects
o Mutated introns
o Mostly no effect (spliced from mature RNA)
o Frameshift mutation = incorrect splicing (e.g. introns spliced into exon sequence)
▪ Abnormal proteins → phenotypic change
▪ Incorrect splicing of -globin mRNA = -thalassemia
Investigate the causes of genetic variation relating to the processes of fertilisation, meiosis &
mutation.
• Increased gene recombination = increased variation between individuals
o Expands gene pool (greater genetic diversity)
▪ More alleles for each gene
▪ More genes for each trait
• Natural selection increases frequency of desired alleles
o Adapted to selection pressures (survival advantage)
o Differential reproductive success = population evolves
Fertilisation • Random fertilisation of genetically unique gametes
o Equal chance of fusion
Meiosis Crossing over
• Non-sister chromatids of homologues exchange corresponding
alleles at chiasma
o Combines alleles from 2 parents = recombinant phenotype
Random segregation
• Alleles of trait separate into different gametes
o 1 allele per gamete
• 50% chance to receive paternal chromosome of certain pair etc.
Independent assortment
• Unique arrangement of homologous pairs on metaphase plate
o 2n (23) possibilities
Mutation • Increases no. of alleles = expands gene pool
o Replication errors during gametogenesis
o Induced by mutagens
Evaluate the effect of mutation, gene flow, & genetic drift on the gene pool of populations.
• Violates Hardy-Weinburg assumptions
o Changes allelic frequency of gene pool
▪ Mutation, migration = expands gene pool
▪ Genetic drift (eg. bottleneck), selective breeding = shrinks gene pool
o Microevolution over generations → speciation
▪ Isolation (eg. geographic) = can’t interbreed
▪ Most prominent effect in small populations
Lili Wang (2019)
Mutations
• Natural selection favours desired mutations
o Retains neutral mutations (evolutionary back-up in changing enviro)
o Eliminates deleterious mutations
Genetic drift
• Alters allele frequencies of gene pool by random chance
• Non-selective: independent of genotype
Bottleneck effect
• Drastically reduces population size (eg. natural disaster)
o Random loss of alleles
• Reduces genetic diversity among cheetahs after mass extinction
Founder effect
• Some individuals geographically isolated from larger population
o No interbreeding with original population = ↓ gene pool
• Increased allelic frequency for retinitis pigmentosa on Tristan da Cunha
Gene flow
• Alters allelic frequency by movement of fertile individuals or gametes
o Migration between populations = ↓ genetic differences
• Interbreeding reduces speciation
o Increases frequency of adaptive alleles by natural selection
Investigate the uses & applications of biotechnology (past, present, future).
• Biotechnology: use of biological materials for human needs
o Includes cheese-making, selective breeding & gene manipulation
• Genetic technology: using genetic material for human needs
• Genetic engineering: intentional manipulation of genetic material for human needs
o Synonymous with genetic manipulation
▪ Sometimes synonymous with genetic modification (which may include exposing to
mutagens)
o Involves recombinant DNA technology
▪ Hepatitis B vaccines, Humulin Selective breeding is usually considered
to be only a biotechnology.
▪ Bt cotton, Golden Rice
▪ Genetically modified bacteria
• Genetically modified organism: genome has been intentionally altered in some way
• Transgenic organism: genome contains DNA from another species
All transgenic organisms are GM (converse is not true).
Lili Wang (2019)
Past biotechnology
Agriculture
• Selective breeding
o Cross-bred organisms with desired traits
o F1 hybrid vigour
▪ Faster genetic improvement = increased productivity
▪ Economically viable (eg. higher milk yield)
o Friesian Jersey = more creamy milk
• Domestication of sheep
o Selectively bred for woolly sheep (6,000 BC)
▪ Unpigmented, longer fleece
▪ Could be shorn
Aboriginal aquaculture
• Harvesting eels (eg. short-finned)
1) System of canals & stone weirs
▪ Selectively funnelled eels to inland areas
2) Collected by Budj Bim eel traps
▪ Favours reproduction of certain eels
▪ Desired traits (eg. more meat)
Classical biotechnology (1800s–mid-1900s)
Fermentation
• Ancient Egypt (6,000 ya):
o Yeast (from spores) in damp areas
o Fermented sugars in milled grain → alcohol
• Pure strains of S. cerevisiae, cultured by Pasteur
o Leavening agent in bread
o Foaming action with gas bubbles (i.e. CO2)
▪ Dough expands, becomes more elastic
Medicine & antibiotic production
• Medicinal plants:
o Salicin (willow bark) – inflammation
o Milk of opium poppy – pain (Egypt)
o Quinine (Jesuit bark) – fever (SA)
• Penicillium fungus
o Produces penicillin (first modern antibiotic)
Lili Wang (2019)
Artificial pollination
• Used by Mendel
o Crossbred pea plants – led to his 3 laws of inheritance (M5)
• Pollen (anther) brushed against stamens
o Pure lines with desired features
o Rust-resistant Federation wheat
Modern biotechnology (1950s–present)
• Refer to recombinant DNA technology (eg. production of Humulin)
Ethical uses of biotechnology
• Ethical use: conforms to society’s standards
o Development of pest-resistant crops (eg. Bt cotton)
o GM bacteria to produce insulin
o Artificial insemination & pollination
• Possibly unethical: gene editing using CRISPR
Future biotechnology
• CRISPR
o Derived from genome-editing system in bacteria
▪ Guide RNA binds to Cas-9 endonuclease & target DNA sequence
▪ gRNA recognises target sequence → Cas-9 snips DNA at desired location
o Devise new treatments for cancer, HIV etc.
o Germline engineering – altering DNA in germ cells or gametes
▪ Changes are passed onto children
▪ Ethical issues about enhancing normal traits (eg. intelligence)
• Gene therapy
o Inserting new & functional genes to replace defective genes
▪ Severe Combined Immunodeficiency (SCID), potentially cystic fibrosis
▪ No impact on germ cells – cannot be inherited
o Allows these individuals to survive to reproductive age
▪ Could increase frequency of affected alleles
▪ Hinders natural selection from eliminating such alleles
Compare the processes & outcomes of reproductive technologies.
Artificial insemination
Process
1) Collect semen from genetically superior bull
o Artificial vagina
o Electro-ejaculation
Lili Wang (2019)
2) Divide into semen straws
o Dilute semen – glycerol, skim milk, fructose & antibiotics
o Frozen under N2 (l) to extend shelf-life
3) Deposit semen in female reproductive tract (on heat)
o Inseminating gun into vagina
o 2⁄3 of semen past cervix
Outcomes
▪ Conservation of endangered species
o Potentially for grey nurse sharks
o Broad-nosed seven-gill shark as model
▪ Breeder manipulates gene pool – not ‘nature’
o Alleles are selected to increase output (i.e. disrupts natural selection)
o Selective breeding of desired phenotypes
▪ Passes on new allelic combinations
Advantages Disadvantages
▪ Faster genetic improvement than natural mating ▪ Expensive: specialised equipment, semen
o Superior genotype to more offspring ▪ Reduces genetic diversity
(eg. more marbling for exports) o Less bulls used
o Efficiently reaches market specs o Express recessive traits (eg. dwarfism)
o Economically viable = $$ o More uniform breeds = genetic erosion
▪ Frozen semen straws ▪ Requires more expertise
o Overseas bloodlines o Detecting oestrus
o Less injury during transport o Damaged semen reduces conception
o Cross-breeding without different bulls
Example Ethical issues
• Toystory – American stud bull • Procreation via natural means
o 500,000 offspring (50 countries) • Animal welfare (eg. pain)
Artificial pollination
Process
1) Remove stamens of flower
2) Dust pollen onto stigma
o Same plant (self-pollination)
o Different plant (cross-pollination)
Outcomes
• Hybridisation: cross different genotypes
o F1 hybrid vigour (eg. aphid-resistant Hunterfield lucerne)
▪ Meiosis (eg. random segregation) = inconsistent F2
Lili Wang (2019)
o Hybrid varieties increase genetic diversity in short-term
▪ Introduces new allelic combinations
▪ Purple Straw 14A × Yandilla = Federation wheat
o Overuse shrinks gene pool: cannot adapt to changing enviro
▪ Texas male sterile cytoplasm in 90% maize crops
▪ Susceptible to Race T fungal gene → Southern corn blight epidemic (1970)
Investigate & assess the effectiveness of cloning.
Whole-organism (reproductive) cloning
Somatic cell nuclear transfer
1) Isolate nucleus from somatic cell (eg. udder cell from sheep 1)
2) Enucleate unfertilised ovum from sheep 2
o Remove nucleus using micropipette
3) Inject udder nucleus into enucleated ovum
o Treated with electric shock
o Fuses into ‘fertilised’ ovum
4) Cell undergoes mitosis (in vitro)
5) Implant embryo into uterus of sheep 3
o Acts as surrogate
6) Embryo is born as a genetic clone to sheep 1
Ethics of cloning
o Prohibition of Cloning Act 2002
o Alters gene pool: plays God
o Breeder controls phenotype
o Reduced uncertainty to reproduce desired traits
Lili Wang (2019)
o Smaller no. of viable parents (shrinks gene pool)
o Susceptible to disease (eg. Dolly’s arthritis)
o Conserves specific genotypes
▪ Identical to 1 parent
▪ Eliminates unselected traits
Effectiveness of SCNT
• Used as therapeutic cloning
o Pluripotent stem cells obtained from SCNT embryo
o Not implanted into mother
• Potential as regenerative medicine (eg. scarring in Crohn’s disease)
o Inject stem cells to repair damaged tissue
Gene cloning
Process
1) Isolate DNA
o Target DNA from donor genome
o Vector DNA (eg. plasmids, BACs)
2) Cut target DNA & plasmid into fragments
o Same restriction enzyme
o Produce complementary ‘sticky ends’
3) Ligation
o Base-pairing between ‘sticky ends’
o DNA ligase forms phosphodiester bond
▪ Seals DNA backbone = forms recombinant plasmid
4) Transformation
o Introduce recombinant plasmid into host cell (eg. E. coli)
▪ Electroporation
• Pores in cell wall
• Increased permeability to DNA molecules
▪ Micro-injection
• Inject DNA into nucleus (eg. ovum)
• If fertilised, transgenic species
▪ Biolistics
• Fire DNA-coated gold particles using ‘gene gun’
▪ Transduction
• Viral vector (eg. adenovirus) delivers DNA
• Injected into bloodstream or aerosol delivery (eg. nasal spray)
o Recombinant DNA replicates as host reproduces (‘in vivo’)
Lili Wang (2019)
5) Test presence of recombinant DNA in host
o Bacteria placed onto antibiotic plate
▪ Without plasmid, bacterium dies
▪ No antibiotic-resistant gene
o Surviving bacteria forms a colony
6) Isolate plasmid from colonies
o Check correct sequence via DNA sequencing or PCR
Investigate the uses & advantages of current genetic technologies that induce genetic change.
• Refer to transgenic species, SCNT, CRISPR & gene therapy
• Focus on genetic engineering
Describe techniques & applications used in recombinant DNA technology.
Hepatitis B vaccines • Surface antigen genes from HBV are inserted into yeast genome
o Yeast produces surface antigen polypeptide
o Incorporated into vaccine & administered
Advantages
• Reduces spread of HBV
• Immunity against HB (esp. newborns)
Disadvantages
• Side effects (eg. pain at injection site)
Genetically modified • Humulin treats both type 1 & 2 diabetes
bacteria 1) Extract plasmids from E. coli
2) Restriction enzyme cuts plasmid at specific restriction sites
Lili Wang (2019)
3) Same restriction enzyme cuts human insulin gene from
chromosome 11 in pancreatic cells
▪ Produces complementary sticky ends
▪ Gene inserts into plasmid = joined by DNA ligase
4) E. coli placed into solution of modified plasmids
▪ Soften cell walls (more readily transformed)
▪ Reproduce in large vats = Humulin!
Faster reproduction = higher yield
Absorbed faster
Less side effects than insulin from pigs/cows
• Gene to produce chymosin inserted into E. coli
o Enzyme produces curd for cheese-making
o Purer than mixture derived from veal calves
• Bioremediation
o Bacteria converts pollutants into less toxic form
o Gene for Hg2+ transport system inserted into E. coli, from
Pseudomonas
Golden Rice • Insertion of 2 genes (soil bacterium, maize or daffodil) into rice genome
• Endosperm produces beta carotene (precursor of vitamin A)
Advantages
• Reduces vit. A deficiency, esp. areas with shortage of dietary vit. A
o Cause of irreversible blindness
Disadvantages
• Earlier version with daffodil gene not as effective
o Poor crop yields
o Reduced beta carotene production
• Opposition from environmental groups (eg. Greenpeace)
o Concerns about cost of development
• Loss of biodiversity
Evaluate the use of biotechnology in agriculture.
• Refer to transgenic species (i.e. Golden Rice & Bt cotton)
Bt cotton
• 3 genes from soil bacterium, Bacillus thuringiensis
• Produces endotoxins toxic to Helicoverpa larvae
o Paralyses digestive tract (cannot feed on cotton crops)
Process
1. Isolate genes that code for Delta endotoxins from Bt genome
o Cut using restriction enzyme
Lili Wang (2019)
2. Same restriction enzyme cuts plasmid of vector bacteria (eg. Ti plasmid of Agrobacterium
tumefaciens)
o Bt genes mix with plasmid
o Forms a continuous loop of DNA
▪ Placed into growth medium to multiply
3. Place cotton plant embryos into solution (contains Agrobacteria with modified Ti plasmid
o Plasmid transferred into cotton nuclei
4. Incorporates Bt genes into cotton genome
Potential benefits Ethical uses
• Cost-effective production • Corporate control of Bt cotton
o Higher yield per plant (eg. wheat) o Expensive = financial debt, additional stress
o -93% insecticide use (since 1997) o Annual purchase of seeds
• Less production losses (eg. Bt cotton) o License fee for Monsanto
• Increased growth (eg. AquAdvantage ▪ Small-scale farmers cannot afford
salmon) ▪ No competitive advantage
o Reaches market weight faster • Genetic proliferation in the wild
o Grown in captivity o Outcompetes non-GM cotton
▪ Growth hormone o Loss of biodiversity
(Chinook salmon) • Conflict with cultural/religious views (playing God)
▪ Promoter sequence (ocean • Gene flow with non-GM cotton = mislabelled
pout) produce
• Biofortified crops (eg. Golden Rice) o Could be mistakenly sold as organic
Research directions
• C4 rice: reduces photorespiration in tropical climates
o Concentrates photosynthesis in specialised leaf cells
o Grown in hotter climates (minimises food shortages)
• CRISPR – corn with larger kernels
Effect on biodiversity
• Within a species,
o Introduces transgenes into gene pool = short-term increase in genetic diversity
o In long-term, reduces genetic diversity
▪ More productive = overuse of transgenic species, possible monocultures
▪ Could be wiped out by selection pressures (eg. disease)
• Between species,
o Interbreeding with non-GM species = could outcompete wild populations
o Possible extinction
Lili Wang (2019)
• On habitat diversity,
o Disturb ecosystems (eg. shorter food chains)
o Toxic to non-target species (eg. Monarch butterflies)
o Less pesticide runoff (eg. Bt cotton) = maintains aquatic life
Evaluate the use of biotechnology in medicine.
• Refer to Hepatitis B vaccines & PGT
Preimplantation genetic testing (PGT)
• Screens embryos (before implantation) or oocytes (before fertilisation)
Process
1) Laser removes cells from embryo (when greater than 8 cells)
o Hole in zona pellucida
o Preferably, day 5
2) PCR amplifies biopsied cells
o Screened for defects using array comparative genomic hybridisation (aCGH)
Advantages
• Performed before pregnancy
o Less terminations compared to chorionic villus sampling
• Detects congenital disorders (eg. chromosomal)
o Selects healthy embryos to implant
▪ High-risk mothers (more trisomy 21 among >35 y.o.)
▪ Carriers (eg. sex-linked)
Risks
• Invasive biopsy: could damage embryo
• False negative results (2%)
o Mosaic syndromes
Ethical concerns
o Select embryo for non-medical reasons (eg. desired sex)
o ‘Saviour sibling’
o Matching HLA type
▪ Stem-cell transplants for sick sibling
▪ Child’s welfare (eg. emotional harm)
Research directions
• Identifies genetic outliers in intelligence
• Screening for polygenic traits (eg. height)
Lili Wang (2019)
Evaluate the use of genetic technologies in industries.
Bioremediation
o Use of microorganisms to break down pollutants
o Enzymatically attacks → harmless products
o Oxidative: oxidises petroleum
o Anaerobic: reduces chloromethanes
o Recombinant E. coli
o Expresses gene for Hg2+ transport system (from Pseudomonas)
o Selectively adsorbs to Hg
Advantages
o More sustainable than remediation alternatives
o ‘Pump & treat’ – pumps out contaminated groundwater
o Thermal desorption – volatilises contaminants (eg. Hg)
o Cost-effective (esp. oil spills)
o Reduces mobility of heavy metals (eg. Bacillus cereus)
o Reduces human exposure
Disadvantages
o Limited to biodegradable pollutants
o Could produce a more toxic end-product
o Specific catalytic conditions (eg. pH)
o Requires pH buffers, adequate nutrition & specialised cultures
Ethical concerns
• Genetic engineering of bacterial DNA
o Interferes with nature
o Horizontal gene transfer: ↓ gene pool
Research directions
• Perform genetic engineering on more bacteria species
• Cd-binding peptides engineered into Pseudomonas
o Increased metal adsorption
o Reduces bioaccumulation in environment
Lili Wang (2019)