How a nucleotide sequence codes for the amino acid
Making the RNA (transcription)
- DNA on gene unwinds and unzips. H bonds between bases break.
- DNA + RNA nucleotides (nuceloplasm) activated contain 2P groups = ATP GTP CTP UTP.
- Binds with template strand A+U C+G (catalysed by RNA polymerase)
- 2P released - provides energy for bonding nucleotides to eachother
- mRNA produced is copy of coding strand of DNA
- mRNA passes out of pore in nuclear envelope to ribosome
Constructing a Polypeptide (translation)
- mRNA binds to ribosome: (2 codons in subunit) (first codon AUG)
- ATP + enzyme help tRNA with methionine + UAG to H bond
- 2nd tRNA binds 2nd subunit peptide bond forms between amino acids
- Ribsome moves along mRNA to 3rd codon and tRNA brings another amino acid - 1st tRNA leaves
- Coding stops at stop codon UAA, UAC, UGA where no tRNA for them.
Lac Operon - Section of DNA within bacterium's DNA
When Lactose absent from growth medium
- regulator gene expressed, repressor protein synthesised - binding sites: (1) lactose (2) operator region
- repressor protein covers promoter region where RNA polymerase attaches
- RNA polymerase cannot bind - not transcribed into mRNA
- no mRNA genes not translated + B-galactosidase + lactose permase not synthesised.
When Lactose added to growth medium
- lactose (inducer) binds to repressor protien - changes shape doesn't bind to operator region - dissociates
- promotor region unblocked-RNA polymerase binds initiates transcription
- operator-repressor-inducer switch. structural genes Z and Y transcripted + translated into B-galactosidase + lactose permase
- E.coli can use lactose permease to make lactose => glucose + galactose
- These sugars can be used in respiration
Apoptosis - Clean and Tidy cell death
Cell programmed death occurs in multicellular organisms. Orderly and tidy cell death. Opposite to necrosis which is untidy and damaging cell death occurs after trauma and releases hydrolytic enzymes.
- Enzymes breaks down cytoskeleton of cell
- cytoplasm becomes dense with organelles tightly packed
- cell surface membrane changes (blebs form)
- Chromatin condenses + nuclear envelope breaks.
- DNA breaks into fragments
- cell breaks into vesicles taken up by phagocytosis
- debris is disposed of and doesn't damage other cells and tissues
- whole process is very quick.
Meiosis 1 - One cell splits into two
- Prophase 1:
- chromatin condenses through supercoiling, chromosomes come together in homologous pairs = bivalent, chiasmata, crossing over takes place, nucleolus diappears, nucelar envelope disintergrates, spindle forms, lasts for days, months or years depending on species and type of gamete.
- Metaphase 1:
- bivalents line up across equator, arranged randomly, chiasmata still present
- Anaphase 1:
- homologous chromosomes pulled apart, centromeres DO NOT divide, chiasmata separate
- Telophase 1:
- two new nuclear evelopes form, one aroung each set of chromosomes (cytokinesis), most plants go straight from anaphase 1 to meiosis 2.
Meiosis 2 - Two cells split further into four
- Prophase 2:
- nuclear envelope has reformed, nucleolus disappears, chromosomes condense and spindles form
- Metaphase 2:
- chromosome arrange on equator of spindle, spindle attached to centromeres, chromatids are randomly assorted
- Anaphase 2:
- centromeres divide chromatids pulled to opposite poles by spindle fibres
- Telophase 2:
- nuclear envelopes reform around daughter cells, animals 2 cells divide into four cells, plants tetrad of 4 haploid cells is formed.
Interactions Between Gene Loci (Epistasis)
- Recessive epistasis:
- two gene loci A/a and B/b involved, homozygous aa is epistatic to both alleles, ratio of 9:3:4
- Dominant epistatsis:
- dominant allele at one gene locus masks expression of another. Ratio of 12:3:1 or 13:3
- complementaty action:
- two gene loci complement each other if one codes for an intermediate and second locus codes for an enzyme which converts intermediate into final pigment. ratio of 9:7
Artifical selection in Cows and Wheat
Modern dairy cow:
- cow's milk yield is measured
- bulls tested to find which ones produce daughters with high milk yield.
- few good quality bulls need to be kept
- elite cows given hormones to produce many eggs
- eggs fertilsed in vitro and implanted into surrogate mothers
- embryos could be cloned
Bread Wheat Triticum aestivum:
- Domestication and artifical selection (AuAu)
- Mutation that doubled chromosome number (AuB)
- Mutation that doubled chromosome number (AuBD)
- Common wheat (AuAuBBDD)
Natural and Artificial Plant Clones
- Elm trees after damage of parent plant grow root suckers (basal sprouts) which grow from trunk and form more trees.
- Disadvantage Dutch Elm Disease.
- Cuttings - section of stem is cut and planted with hormones
- Grafting - section of plant joined to already growing stem
- small piece of tissue taken from plant (explant)
- placed on nutrient growth medium
- cells divide and form undifferentiated cells (callus)
- after few weeks callus removed and placed on growing medium for shoot growth
- transferred onto medium containing hormones for root growth
- growing plants transferred to greenhouse before planting outside.
Natural and Artificial Animal Clones
- Identical twins when zygote separates into two inside uterus at early development means organisms are clones
- Splitting embryos
- in vitro fertilisation
- grown till 16 cells
- split and implanted into surrogate mothers
- Nuclear transfer
- remove mammary cells and extract nucleus
- extract ovum cells and remove nucleus
- electro-fusion mammary nucleus with enucleate ovum
- Culture in oviduct of sheep
- Implant in surrogate mother's uterus
Standard Growth Curve for a Culture of Microorgani
- Lag Phase
- adjusting to surroundings
- cells are active but not reproducing
- population fairly constant
- length depends on growing conditions
- Log (exponential) phase
- population size doubles each generation
- length depends on how quick organisms reproduce
- Stationary Phase
- nutrient levels decrease, waste products + metabolites increase
- organisms die at same time new individuals are being produced
- carrying capacity
- Decline or Death Phase
- nutrient exhaustion
- increased levels of toxic waste products + metabolites
- death rate increases over reproduction
- All organisms die in closed system
Method for Immobilising Enzymes
- Immobilising support binds to enzymes (glass beads/clays/resins)
- hydrophobic interactions and ionic links
- can become detached (leakage)
- Covalent Bonding
- covalently bonded to support (clay particles)
- cross linking agent gluteraldehyde or sepharose
- binding strong, little leakage, doesn't immobilise large amounts
- trapped in gel bead or network of cellulose fibres
- reaction rates can be reduced, active site less easily available
- Membrane separation
- physically separated by partially permeable membrane
- substrate molecules small to pass through membrane
- product molecules small to pass back
Sequencing the genome of an organism
- genomes are mapped (location of micro-satellites help in this)
- 'shotgun' approach - genome is sheared into small sections (100,000 base pairs
- sections placed into bacterial artificial chromosomes (BACs) and transferred to E.coli. and are grown in a culture (clone libraries)
In order to sequence BAC section:
- cells containing specific BACs are taken and cultured. DNA extracted using restriction enzymes.
- fragments separated using electrophoresis
- each fragment is sequenced
- computer programme compares overlapping regions from cuts made by enzymes and reassembles whole BAC segment sequence.
- DNA samples treated with restriction enzymes (cuts them into fragments)
- DNA samples placed into wells cut into gel.
- gel immersed in buffer solution and current passes through for fixed time (2 hours)
- DNA negative because of phophoryl groups (attracted to positive end) so it diffuses through gel
- shorter DNA strands move faster than longer ones
- position of fragments shown by using dye that stains DNA
- nylon/nitrocellulose sheet placed over gel and blotted
- DNA fragments transferred to sheet can be analysed
Polymerase Chain reaction - PCR
- DNA mixed with DNA nucleotides and DNA polymerase
- heated to 95 degrees breaks H bonds (becomes single stranded)
- primers added (DNA strands 10-20 bases)
- temperature reduced to 55 degrees primer bind to DNA (some double stranded DNA)
- DNA polymerase binds to these sections
- temperature raised to 72 degrees - optimum temp for DNA polymerase (free nucleotides bind to unwound DNA)
- DNA polymerase reaches end of DNA strand and new DNA is formed
- repeated many times so DNA increases exponentially
Automated DNA sequencing - with fluorescent marker
- primer anneals at 3' end of template strand - DNA polymerase attaches
- free nucleotides bind
- modified nucleotides (with fluorescent markers which when they bind throw off the sequence so that the strand cannot bind more nucleotides)
- Many DNA molecules are made, fragments vary in size, the final nucleotide is tagged with a colour
- strands run through a machine and laser reads colour sequence with strands varying from only a single nucleotide attached then 2, then 3, then 4 so the sequence of colours is the sequence of bases.
How Restriction Enzymes and Ligases Work
- restriction enzymes cut DNA at a specific point (where a base sequence occurs at the restriction site) uses hydrolysis
- this leaves some exposed bases called a sticky end
- DNA ligase catalyses condensation reaction and seals DNA nucleotides together.
- to join DNA fragments from 2 different sources they need to be cut with the same restriction enzymes so the sticky ends are complementary and bases pair up.
- results in recombinant DNA.
- Obtaining gene to be engineered
- mRNA used as template to make copy of gene
- automated polynucleotide sequencer
- DNA probe used to locate DNA fragments-cut with restriction enzymes
- Placing DNA in vector
- sealed in bacterial plasmid using ligase
- sealed into virus or yeast cells
- vectors contain regulatory sequences of DNA so inserted DNA is transcribed.
- Getting gene into recipient
- Electroporation: high voltage pulse applied to disrupt membrane
- Micro injection: DNA injected using fine micropippete into host nucleus
- Viral transfer: virus inserts DNA directly
- Ti Plasmids: inserted into soil bacterium and plants can be infected with it so DNA plasmid is in plant's genome
- Liposomes: DNA wrapped in lipids, can cross lipid membrane by diffusion.
Bacterial Conjugation + Engineering to Produce Ins
- conjugation tube forms between donor and recipient, enzyme makes nick in plasmid
- plasmid DNA replication starts, free strand moves through tube
- recipient cell replication starts
- cells move apart and plasmid in each forms a circle
- transcription of pancreas cells to give mRNA which is isolated + treated with reverse transcriptase, treatment with DNA polymerase produces cDNA strands which are copies of human insulin gene, sticky ends complementary to those of plasmid are added
- plasmid removed from E.Coli and cur open by restriction enzyme
- 2 pieces are splices together and sealed using DNA ligase
- plasmid containing human insulin gene inserted into E.Coli cell
- Transgenic bacteria capable of producing human insulin is generated.
Identifying transformed bacteria
Plasmid vectors with genetic markers:
- E.Coli plasmids are resistant to ampicillin and tetracycline
- plasmids are cut by restriction enzyme that has its restriction site in the middle of tetracyclineresistance.This means that plasmids taking up the gene won't be resistant. Ampicllin resistance still works.
Replica plating is then used:
- bacteria cells grown on nutrient agar to form colonies
- these cells are transferred onto agar that have been made with ampicilin
- some are transferred onto agar that have been made with tetracycline
- by keeping track of which has grown on which we know that the colonies on ampicillin agar but not on tetracycline agar must have taken up plasmid with insulin gene.
- these can be grown on a large scale for insulin production for example.
GM Golden Rice
- precursor molecules
- Phytoene synthase from daffodil
- Crt 1 enzyme from bacterium
- enzymes already exist in rice endosperm
- Beta carotene
- Precursor molecule which is converted to active vitamin A in the gut.
Primary Succession and Ecosystem Surveying
Dune into a woodland
- pioneer plants like sea rocket + prickly sandwort colonise above water mark
- mini dunes formed, nutrients accumulate, bigger plants sea sandwort colonise
- sea spurge grows and marram grass
- as nutrients build up other plant colonise
- bacteria root nodules convert nitrogen into nitrates
- this continues until climax community is reached
Surveying an ecosystem:
- line transect: regular intervals make a note of species touching tape
- belt transects: regular intervals place quadrat next to line and studying each
- Quadrats: presence or absence of species (abundance) or estimate count of individuals (abundance)
- point quadrat: point to ground and lower each needle and record the species that tip touches on its way down.
Predator-Prey Realtionship and Timber Production
Predators and Prey
- when predatory pop gets bigger more prey are eaten
- prey pop gets smaller less food for predators
- less food fewer predators can survive and population size reduces
- fewer predators, fewer prey, population size increase
- more prey, predator pop gets bigger, cycle continues
Sustainable Timber production
- Tree replacement with new ones
- even extraction of timber
- local people should derive benefit
- Coppicing: cutting trunk of deciduous tree close to the ground
- Pollarding: cutting tree higher up.
Photo-tropism and Shedding Leaves
- bends towards light source
- shaded side elongates faster than illuminated side
- auxins transported to shaded side and promote increase in rate of elongation
- leaf senescence causes auxin production at tip of leaf to drop
- cells in abscission zone more sensitive to ethene
- drop in auxin concentration causes ethene production
- produces enzyme cellulase which digests walls of cells in abscission zone
- separates petiole from stem
- when apex of plant is cut off lateral bud growth increases
- auxin concentration in lateral buds increase
- abscisic acid (inhibits growth) levels kept high normally in lateral buds because of high concentration of auxin in apex
- when tip removed abscisic acid concentrations drop and buds grow
- applying cytokinins to buds overrides apical dominance
- shoot apex is sink for cytokinins produced in roots, when it is removed cytokinin spread more evenly promoting growth in buds.
Gibberellin and stem elongation:
- tall pea plants have higher concentration of gibberellin than small pea plants
- by grafting a plant which has no gibberellin production onto one that can it grows tall because it uses the precursor molecule GA20 to use with its enzyme to make GA1
- This shows GA1 is the hormone responsible for stem elongation
The organisation and roles of the nervous system
- Central nervous system: consists of brain and spinal cord made up of grey and white matter
- Peripheral nervous system: made of neurones that carry impuses into and out of CNS.
- Somatic nervous system: carries impulses from CNS to skeletal muscles, which are under conscious control
- Autonomic nervous system: carries impulses from CNS to cardiac muscle to smooth muscle in the gut wall and to glands which are not under voluntary control. most neurones unmyleinated, swells called ganglions.
- Sympathetic subsystem: most active in stress, pre ganglionic neurones short, secretes noradrenaline
- Parasympathetic subsystem: most active in relaxation, pre ganglionic neurones vary in length, secrete acetylcholine
Neuromuscular junction and power stroke
- impulses arrive and vesicles fuse with pre-synaptic membrane
- acetyl choline is releases and binds to receptors on muscle fibre (sarcolemma) causing depolarisation
- Depolarisations travels down T system (tubules)
- T system leads to Ca2+ release from sarcoplasmic reticulum
- Ca2+ binds to protein in muscle leading to contraction
- acetyl-cholinesterase breaks down acetyl-choline so that contractions only occurs when impulses arrive continuously.
- Myosin head attached to actin filaments and forms cross-bridge
- head group bends causing thin filament to be pulled along and so overlap more with thick filament (power stroke - ADP + Pi)
- cross bridge is broken as new ATP attaches to myosin head
- head moves back as ATP is hydrolysed to ADP + Pi, can then form cross-bridge with thin filament further along and bend again.