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203 Cards in this Set
- Front
- Back
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What is a prokaryote? |
An organism that is comprised of a cell which does not have a nucleus. Includes bacteria and archaea. |
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What is a eukaryote? |
An organism comprised of a cell/cells which have a nucleus and a nuclear membrane. Includes protists, fungi, animals and plants. |
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How do prokaryotes store genetic material? |
Stores genetic material as a single circular DNA molecule which is condensed to form a nucleoid which is free in the cytoplasm. Plasmids are additional rings of DNA in the cytoplasm which can be exchanged with other bacteria. |
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How do eukaryotes store genetic material? |
Store DNA in the nucleus. Genetic material is linear DNA organised as proper chromosomes. DNA is wrapped around histone proteins to form nucleosomes. |
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What is a flagellum? |
A tail used in prokaryotic organisms for motion. |
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What are pili? |
Surface projections on prokaryotic cells that help them attach to surfaces and can be used to exchange plasmids with other bacteria. |
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What is the capsule? |
Secretion on the surface on prokaryotic cells made of mucilage that helps the cell adhere to surfaces. |
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What is significant about the bacterial cell wall? |
It is made of peptidoglycan, protects the cell, helps it to maintain shape and prevents excessive uptake of water. |
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What is the cytoskeleton? |
System of fibres in a eukaryotic organism that provide support/movement/organisation of organelles. |
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What is cytosol? |
Liquid component of cytoplasm in eukaryotic cells. |
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What are endomembranes? |
Membranes that compartmentalise different parts of the metabolism. They make up the endoplasmic reticulum and the Golgi apparatus. |
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What is the Golgi apparatus? |
Organelle involved in the packaging of complex molecules into vesicles. |
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What are ribosomes? |
Site of protein synthesis; they are larger in eukaryotic organisms. |
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What is the endoplasmic reticulum? |
Involved in the process of protein synthesis and packaging. |
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What are micro bodies? |
Have a single membrane around them and are produced through budding from the ER or Golgi body. |
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What are mitochondria? |
Site of aerobic respiration in eukaryotic organisms. |
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What are centrioles? |
Used during division in animal cells. Formed by the centrosome which is also known as the micro tubule organising centre. |
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What are micro villi? |
Membrane extensions in animal cells that greatly increase the surface area for absorption (ie in the small intestine). |
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What are lysosomes? |
Specialised vesicles which digest substances by attaching to them and releasing digestive enzymes. |
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What is significant about the MTOCs in plant cells? |
They have more than one MTOC and these are distributed close to the nucleus. |
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What are chloroplasts? |
Centres of photosynthesis. They have a double membrane. |
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What is the central sap vacuole? |
Stores sugars, ions and pigments in plant cells. |
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What is the cell wall? |
Maintains plant cell shape. Made of cellulose. |
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What are plasmodemata? |
Cytoplasmic links between plant cells. |
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What is the middle lamella? |
Glues plant cell walls together, made of pectin. |
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What are the substages in interphase? |
G1 - first growth stage. Cell makes new proteins and copies of organelles. S - DNA replication occurs. G2 - growth stage: cell makes more proteins and copies organelles in preparation for mitosis. |
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What are the checkpoints in interphase? |
G1 checkpoint - near end of G1. Cell size is monitored. G2 checkpoint - at the end of G2. Checks DNA replication success. Can only be passed if mitosis promoting factor is present. M checkpoint - during metaphase. Monitors chromosome alignment. |
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What is Mitosis Promoting Factor (MPF)? |
A protein complex controlling the entry of cells into mitosis. |
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What happens during prophase? |
Chromosomes condense and spindle fibres attach to them at centromeres. The nuclear membrane disintegrates. |
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What happens during metaphase? |
The spindle fibres more towards the chromosomes so they line up on the metaphase plate at the equator of the cell. |
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What happens during anaphase? |
Spindle fibres pull chromatids apart. |
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What happens during telophase? |
The separated chromosomes are pulled to opposite poles to form daughter nuclei. Chromosomes start to uncoil and the nuclear membrane is made again. |
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What happens during cytokinesis? |
The membrane is pulled in by part of the cytoskeleton to form two daughter cells. Plant cells have to form a middle lamella and cell wall before the membrane is made. |
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What is the mitotic index? |
The percentage of cells undergoing mitosis in a sample. |
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Describe normal differentiation? |
Cells with different structures and functions are specialised for different roles. These are grouped into tissues, and these tissues are grouped into organs. |
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What are the requirements for gene switching in the lac operon mechanism? |
The system must have: -A regulator gene which codes for a repressor molecule. -The repressor molecule which binds the operator to the inducer. -A structural gene that makes beta galactosidase, an enzyme which breaks down lactose. -An operator consisting of a length of DNA 'upstream' of the structural gene. -An inducer molecule (lactose) which binds to the repressor molecule. |
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What happens when there is no lactose present for the E.coli? |
1) The regulator gene makes the repressor molecule. 2) This attaches to the operator so the transcription enzyme cannot read the structural gene. 3) No Beta Galactosidase is made and the bacterium does not waste energy making an enzyme it doesn't need. |
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What happens when there is lactose present for the E.coli? |
Lactose acts as the inducer to cause the production of Beta Galactosidase. The regulator still makes the repressor molecule, but now the inducer attaches to it. This leaves the operator open so that the transciption enzyme can read the structural gene; Beta Galactosidase is produced and lactose can be broken down. |
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What are proliferation genes? |
Genes that code for proteins which promote cell division only when an external signal is received, for example, when a wound must be repaired. |
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What are proliferation genes also known as, and why? |
Proto-oncogenes. When they mutate, they form oncogenes, which are found in many cancers. These mutations give proteins with abnormal shapes which stimulate excessive cell division and tumour formation, even in the absence of any external signal. |
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How else can proliferation genes be considered in terms of alleles? |
Oncogenes can be thought of dominant alleles, because the mutation only has to happen in one gene of the pair. |
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What are anti-proliferation genes? |
Genes which code for proteins which restrict cell division by operating at the cell cycle checkpoints. |
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What are anti-proliferation genes also known as and why? |
Tumour suppressor genes. They normally prevent excessive cell division. |
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How can the cancer causing mutations to anti-proliferation genes be considered in terms of alleles? |
One functioning can still produce the protein to inhibit the cell cycle, so both copies of the gene must mutate before the control of the cell sycle is lost and a tumour starts forming. For this reason, they can be thought of as recessive. |
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What do mammalian cell cultures require? |
-Aseptic conditions -Solid surface -Growth factors + nutrients -In a complex growth media
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Name an example of a growth factor. Why are they used? |
Foetal bovine serum (FBS) promotes cell proliferation. |
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Why are antibiotics used? |
To prevent bacterial contamination. |
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Name an example of a proteolytic enzyme. Why is it used in cell culture? |
Trypsin is used to detach the cells from the source tissue. |
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Describe the process of subculturing. |
Cells adhere to the surface of the agar, then spread out and divide until they form a monolayer and become confluent. Cells soon use up all the nutrients in the medium so they must be subcultured into a fresh flask by detaching them from the agar with proteolytic enzymes again. |
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Why is it difficult to maintain cultures of mammalian cells? |
Cells die out after a finite number (about 60, the Hayflick limit) of divisions in culture. |
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In what kind of cells does the Hayflick limit not exist? |
Immortal cell lines, such as cancer cells, stem cells, or cells which undergo a genetic change to make them immortal. |
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What do plant cell cultures require? |
-Macronutrients, micronutrients, carbon sources and vitamins -Growth regulators like cytokinins and auxins -Aseptic conditions -Suitable growth medium |
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What are explants? |
Small pieces of plant tissue that are placed on a solid medium to either promote shoot growth or callus growth. Organ formation is then stimulated by altering the rate of cytokinins (to promote shoot growth) and auxins (to promote root growth). |
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How are protoplasts created? |
Cell walls are removed from plant cells with the enzymes pectinase and cellulase. The resulting cells can be grown in liquid medium with a cell suspension culture and treated with growth regulators to induce embryogenesis to generate whole new embryonic plants. Protoplasts can also be encouraged to form a callus, as with explants. |
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What is the purpose of plant cell tissue culture? |
Plant cell suspension cultures allow screening for beneficial traits such as heat or salinity tolerance or the selection of virus free cells for cloning. Can be used to harvest useful cell secretions. |
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What is micropropagation? |
The generation of many cloned plantlets from one source plant. Allows rapid upscaling to field trials. |
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What is glucose? |
The monosaccharide which forms the building block for many polysaccharides. |
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What are the two structure of glucose? |
The 'linear' form, which is actually more of a C-shape due to the 3D angles of the chemical bonds with the of of 5' near the O of 1'.
The ring form. If the OH is below the ring it is alpha glucose, if it is above the ring it is beta glucose. |
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What are the relative abundances of the two forms of glucose? |
The two forms exist in equilibrium but this equilibrium is skewed towards the ring form. |
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How are monomers linked together? |
By enzyme catalysed reactions that cause a condensation reaction between the OH groups of carbon 1 and 4. Monomers of alpha glucose are linked by alpha 1,4 glycosydic bonds and monomers of beta glucose are linked by beta 1,4 glycosidic bonds. |
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Describe the structures of the two different types of starch. |
Both are composed of 1000s of glucose monomers.
Amylose: Long linear molecule with alpha 1,4 glycosidic bonds. No side branches.Long unbranched helix.
Amylopectin: Side branches every 25-30 units, which are linked to the main chain by alpha 1,6 glycosidic bonds. |
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Describe the structure of glycogen. |
Glucose monomers are linked with alpha 1,4 glycosidic bonds with alpha 1,6 glycosidic bond side branches every 10-12 units. |
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Describe the structure of cellulose. |
Thousands of glucose monomers are joined by beta 1,4 glycosidic bonds. Every alternate glucose unit is inverted producing a rigid straight chain with no branches.
Hydrogen bonds form between cellulose chains and groups of about 40 of these chains are cross linked to form fibrils which, in layers, form the cell wall. |
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Why do organisms build storage polysaccharides? |
High concentrations of glucose draw water into the cell, leading to issues for it. Starch and glycogen are insoluble so osmotic issues are avoided. |
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Describe the structure of triglycerides. |
Triglycerides are made up of 3 fatty acids and a glycerol. Glycerol has 3 carbons, each one with an -OH group attached. Fatty acids have a -COOH group and a long hydrocarbon tail at the other. Saturated fatty acids have no double bonds in their tails, while unsaturated ones do. |
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How are glycerol and fatty acids linked? |
Condensation reactions between the -OH of the acid and the -OH of the glycerol, resulting in an ester bond. |
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Describe the structure of phospholipids. |
Similar to triglycerides, but have a phosphate group in place of one of the fatty acids. One fatty acid is saturated, the other is not. Charged groups such as choline may be attached to the phosphate.
The hydrophilic head of the phosphate is comprised of the phosphate and choline groups. The hydrophobic tail is the fatty acids. |
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What do phospholipids have a tendency to do and why? |
Form a bilayer because the hydrophilic heads stay in contact with aqueous solutions (cytosol or extracellular fluids) and the hydrophobic tails stay in the middle of the bilayer, away from water. |
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How is the bilayer kept fluid by the structure of phospholipids? |
Unsaturated fatty acids prevent the phospholipids from packing too closely together and thus keep the membrane fluid. |
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Describe the structure of steroids. |
4 ring structure of 17 carbons. Side chains give different steroids. |
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Why are steroids hydrophobic? |
So they can diffuse across membranes and bind to receptors inside the cell. |
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Describe the structure of cholesterol. |
Cholesterol has an -OH and a -C8H17 side chain. |
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Describe the structure of testosterone. |
Testosterone has a -C=O and an -OH side chain. |
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What is the function of starch? |
Energy storage. |
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What is the function of glycogen? |
Energy storage. |
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What is the function of glucose? |
Broken down in respiration to provide chemical energy. |
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What is the function of cellulose? |
Structural carbohydrate in cell walls. |
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What are the functions of triglycerides? |
Long term energy storage and thermal insulation. |
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What is the function of phospholipids? |
Structural, part of the plasma membrane. |
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What are the functions of steroids? |
Hormonal and structural. |
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What are the functions of different types of proteins? |
Catalytic: enzymes such as DNA polymerase.
Structural: tubulin in the cytoskeleton or collagen in bone and cartilage.
Messenger: Hormones such as ADH and insulin.
Carriers: Membrane proteins such as aquaporin. |
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Describe the structure of amino acids. |
Central carbon atom with an -H group, an -NH2 group, and an -R group attached. |
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What are the four classes of amino acid? |
Acidic, basic, polar and non-polar. All are hydrophilic except non-polar. |
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How are amino acids linked? |
They are linked during translation of mRNA at the ribosome by a condensation reaction that results in a peptide bond. |
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What is the primary structure of proteins? |
The sequence if amino acids that make up a polypeptide chain. The chain has an N terminus at one end and a C terminus at the other. |
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What is the secondary structure of proteins? |
Stabilised by H bonds between the atoms of different peptide bonds in the chain.
Alpha helices - spirals with R groups sticking outwards. Beta sheets - parts of the polypeptide chain running alongside eachother with the R groups sitting above and below. |
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What is the tertiary structure of proteins? |
The overall 3D shape of the protein. Stabilised by interactions between R groups. The incorporation of non-protein prosthetic groups also occurs at this level. |
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What is the quaternary structure of proteins? |
The bonding between the R groups of multiple polypeptide sub-units. |
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Name an example of a protein with quaternary structure. |
Haemoglobin is made up for four sub-units, each with a prosthetic haem group. |
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Describe hydrophobic interactions between R groups. |
Non-polar R groups are mostly arranged to the inside of the protein. The polar, acidic and basic R groups are mostly arranged on the outside. |
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Describe Van der Waal's interactions between R groups. |
Very weak attractions between the electron clouds of atoms. |
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Describe hydrogen bonding between R groups. |
The weak negative charge of the oxygen of C=O is attracted to the weak positive charge on a hydrogen of an OH or NH2 group. |
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Describe ionic bonding between R groups. |
The COOH and NH2 groups ionise to become COO- and NH3+. These groups are strongly charged and attract eachother. |
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Describe disulphide bridges between R groups. |
Covalent bonds form due to reactions between the sulphur containing R groups of cysteines. |
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Describe the structure of nucleotides. |
Phosphate, sugar, and nitrogenous base. RNA sugar is ribose, and DNA sugar is deoxyribose. |
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Name and describe the two different types of bases found in nucleotides. |
Purines: Adenine and guanine, have a double ring structure.
Pyrimidines: Cytosine, thymine and uracil, have a single ring structure. |
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What is the double helix? |
Two strands of nucleotides which are twisted to form a twin spiral. |
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Describe the structure of the double helix. |
Each strand has a backbone made of deoxyribose sugars linked to phosphates by phosphodiester bonds linking carbon 5' to carbon 3' of the next nucleotide. The two strands are antiparallel. |
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How are base pairs linked? |
A - T: Two hydrogen bonds. C - G: Three hydrogen bonds. |
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Describe the structure of RNA |
In RNA the nucleotide also has 5' and 3' at opposite ends. The strand may be looped back on itself to form base paired regions, but it is still a single strand of nucleotides. |
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How is nucleic acid synthesised? |
Synthesis of a new strand of nucleic acid can only happen in the 5' to 3' direction because the polymerase enzyme can only add nucleotides on the 3' end. RNA polymerase builds a 5' to 3' messenger RNA strand because only one strand is required, but DNA requires both strand to be copied (5' to 3' and 3' to 5'). |
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How does DNA replication copy both strands? |
1) Enzymes open up a replication fork in the DNA double helix. 2) The leading strand is synthesised complete in the 5' to 3' direction. 3) Replication starts with the synthesis of a short RNA primer. 4) DNA polymerase starts to build the new chain and then catalyses the formation of the phosphodiester bond between the end of the growing strand and the new nucleotide. 5) The other strand (lagging strand) is made in short 5' to 3' fragments by DNA polymerase. 6) After the RNA primer is replaced with DNA, DNA ligase links the strands together by catalysing the formation of phosphodiester bonds. |
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Describe the structure of the phospholipid bilayer. |
Hydrophobic tails are in the middle of the bilayer and hydrophilic heads are on the outside.
The hydrophilic centre forms a barrier to the passage of polar molecules and ions. |
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How is membrane fluidity controlled. |
The structure of cholesterol, with its hydrophilic head and hydrophobic tail, reduces membrane fluidity and prevents lipid crystallisation at low temperatures. |
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What are glycoproteins and glycolipids? |
Proteins and phospholipids that have carbohydrate chains added to them and exist on the outside of the membrane. |
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What are the two main types of proteins in the protein mosaic? |
Peripheral: Held in place at the surface by charged or polar amino acid R groups.
Integral: Held firmly in place with strong hydrophobic interactions with the lipid tails. Either transmembrane or embedded in one side of the bilayer only. |
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What are passive transport proteins? |
Transmembrane proteins that transport molecules down a concentration gradient. Channel proteins provide a pore that cane facilitate or speed up diffusion (eg. aquaporin and water) while carrier proteins can bind to a specific molecule to allow its passage. |
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What are active transport proteins? |
Proteins that pump molecules against the concentration gradient. |
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How is energy provided for active transport and what is it used for? |
Hydrolysis of ATP provides the energy source for the phosphorylation and the conformational change of protein pumps. |
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What is the function of membrane bound enzymes? |
Allow the location of catalysis to be carefully controlled in a cell. |
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What is the function of receptor proteins? |
When signalled, these transmembrane proteins stimulate a response within the cell. |
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What is the function of attachment proteins? |
Provide cytoskeleton attachment points within the membrane for the structural support of the cell. Other proteins attach to the extra cellular matrix to hold the cell in place. In multicellular organisms, proteins form intercellular junctions that provide anchorage to othr cells and hold cells together in tissues. |
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How is cell-cell recognition achieved? |
Through the presence of membrane glycoproteins which can be recognised by other cells. |
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Name an example of how cell-cell recognition is used. |
In the ABO bloodgroup system, recognition is based on the different carbohydrate chains presented by the glycoproteins on the membrane surfaces of red blood cells. |
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What is the sodium potassium pump? |
A transmembrane ATPase found in animal cells that pumps sodium ions out of cells and potassium ions into cells against a concentration gradient. |
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How does the sodium potassium pump work? |
1) In one conformation state, the pump has a high affinity for Na ions. It expose three Na binding sites to the cytosol. Three Na move in and bind to these sites. 2) Phosphorylation causes a conformational change to the protein. 3) The second conformation has a lower affinity for Na, and thus the 3 Na ions are pumped out of the cell. 4) The second conformation has a higher affinity for K ions so two K ions attach to the binding sites. 5) The dephosphorylation restores the protein to its original conformation. 6) The original conformation has a low low affinity to K ions so it releases the two K ions and the cycle repeats. |
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What is the function of the cytoskeleton? |
Cytoskeleton is a network of protein fibres. It provides mechanical support to the cell and it is involved in the movement of cellular components.
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What are microtubules? |
Polymers of a dimer made from alpha tubulin and beta tubulin. Originate from the MTOC, which contains the centrosome. In animals, microtubules are synthesised in the centrioles in the centrosome. |
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What is the role of microtubules in cell division? |
Spindle fibres are made of microtubules. Microtubules attach to proteins at the centromeres of chromatids. Microtubule severing separates the chromatids. |
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How do enzymes work? |
By binding to their substrate and lowering the activation energy to make a reaction happen. |
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What are the different types of enzyme reaction? |
Synthesis: Join molecules together, usually by a condensation reaction where a water molecule is removed.
Degradation: break molecules apart, often by a hydrolysis reaction. |
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What is the induced fit hypothesis? |
The shape of the active site complements that of the substrate, but the active site also has amino acids with an affinity for areas on the substrate molecule. The active site and the substrate bind my forming hydrogen and ionic bonds. The arrival of the substrate brings about a conformational change in the enzyme that allows an induced fit to occur.
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What is the action of proteases? |
Hydrolysis of peptide bonds to break down proteins (eg. pepsin and trypsin from the digestive system bromelain from pineapples) |
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What is the action of nucleases? |
Hydrolysis of phosphodiester bonds to break down nucleic acids (eg. EcoRI used in genetic engineering to cut DNA) |
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What is the action of ATPases? |
Hydrolysis of the phosphoester bond in ATP to form ADP and phosphate (eg. the sodium potassium pump) |
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What is the action of kinases? |
Condensation reaction to add a phosphate group to another molecule (eg. a kinase adds a phosphate to inactive glycogen synthase) |
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How do competitive inhibitors work? |
It is similar to the substrate and binds to the active site of the enzyme impermanently. Increasing substrate conc. eventually dilutes the competitive inhibitor so much that the enzyme molecules begin to bind to the genuine substrate again. The max rate of reaction remains the same. |
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How do non-competitive inhibitors work? |
Binds to a part of the enzyme other than the active site and changes the shape of the active site so that the original substrate can no longer fit, permanently. Max rade of reaction is reduced. |
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What is covalent modification? |
The activation or inactivation of an enzyme by making or breaking covalent bonds. |
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How can kinase enzymes activate or deactivate enzymes? |
By phosphorylating them, adding a phosphate via a condensation reaction. |
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How can phosphatase enzymes activate or deactivate enzymes? |
By dephosphorylating them via a hydrolysis reaction. |
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Name and describe an example of covalent modification. |
Pancreas cells make an inactive from of trypsin called trypsinogen. It is secreted into the small intestine where it is cut by a protease enzyme to form the active trypsin. |
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Why do pancreas cells make inactive trypsinogen? |
Active trypsin may digest the pancreas. In the small intestine this cannot happen due to the small intestine's protective mucus lining. |
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What do modulators do? |
Change the shape of the enzyme by binding to the allosteric site, affecting the enzyme's affinity to its substrate. |
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What do positive modulators (activators) do? |
Bind to the allosteric site and increase the enzyme's affinity to the substrate. |
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What do negative modulators (inhibitors) do? |
Bind to a different allosteric site and reduce the enzyme's affinity for its substrate. |
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How are negative modulators different from non-competitive inhibitors? |
Negative modulators do not bind permanently. |
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What is end product inhibition? |
When the product of a final reaction in a metabolic pathway slows down the first enzyme of the pathway and so slows down its own synthesis. In some cases it acts as a competitive inhibitor, whereas in others it acts as a negative modulator. |
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What are extracellular signalling molecules? |
Molecules from outside a cell which the cell can detect and then respond to. |
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How do hormones work as extracellular signalling molecules? |
They are secreted by one tissue into the blood, then they circulate the bloodstream until they reach their target receptor or are broken down. |
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What are the two categories of signalling mechanisms for hormones? |
Hydrophilic: Peptide hormones such as ADH, insulin and growth hormone. Cannot pass through membranes.
Hydrophobic: Steroid hormones such as testosterone. Can pass through membrane. |
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How does hydrophobic extracellular signalling work? |
They diffuse across the plasma membrane of the target cell and activate gene regulatory proteins, which regulate the transcription of specific genes. |
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How does hydrophilic extracellular signalling work? |
They activate receptor proteins on the surface of a cell. The receptors act as transducers, converting the extracellular binding event into intracellular signals which alter the behaviour of the target cell. |
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Describe and name examples of neurotransmitters. |
Neurotransmitters such as acetylcholine and noradrenaline are hydrophilic peptides. |
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What do polymerases do? |
Catalyse the condensation synthesis of DNA and RNA nucleic acid polymers by catalysing the formation of a phosphodiester bond between a nucleotide and a nucleic acid of the newly synthesised strand. |
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What are primers? |
Short sequences of polymer complementary to the start of the template strand that are required to initiate polymerisation. |
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What does ligase do? |
Catalyse the condensation synthesis of phosphodiester bonds between two strands of nucleic acid polymer. In DNA replication, it joins the sugar-phosphate backbone between fragments of DNA as they are synthesised on the lagging strand. |
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What do endonucleases do? |
Catalyse the hydrolytic degradation of phosphodiester bonds. In digestion, they catalyse the complete breakdown of nucleic acid polymers into nucleotide monomers. |
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What are restriction endonucleases? |
They also break the phosphodiester bonds between adjacent nucleotides in a sequence but they are restricted to making this cut between a specific sequence of nucleotide bases. |
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What s gel elecrophoresis? |
After DNA is digested by a restriction enzyme, fragments of different lengths are produced. These can be separated using gel electrophoresis, where the DNA sample is placed into a well in a porous polysaccharide gel immersed in a buffer solution. A current runs between two electrodes placed in the buffer. DNA is -vely charged so goes towards the anode, with shorter fragments travelling faster and further. This separates the DNA fragments into bands. |
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How is DNA denatured? |
The hydrogen bonds holding the complementary bases together can be broken by heating the DNA to approximately 95 degrees celsius. This results in single stranded DNA. |
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How is DNA annealed? |
When the temperature is lowered, complementary single-stranded sequences form hydrogen bonds to remake a double helix. This formation of a double stranded molecule from single strands is also known as hybridising DNA. |
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What is DNA blotting used for? |
Making an accurate record of the final positions of DNA fragments by drawing the DNA fragments out of the gel and onto a nitrocellulose or nylon filter. |
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What are DNA probes? |
Short, single-stranded sequences of DNA that have been labelled either radioactively or with a fluorescent tag. Probes bind with any complementary sequences of DNA in a sample. |
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What are single locus probes? |
Those which have a complementary sequence at only one location within the genome being probed. Used for paternity tests and for screening for mutations within the gene sequence. |
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What are multi locus probes? |
Those which have a complementary sequence that occurs at many points within the genome being probed. If a genome is digested using restriction enzymes, a multi locus probe will anneal at many locations and produce a pattern of bands known as a DNA profile. |
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What are the initial requirements for the polymerase chain reaction (PCR)? |
-Template DNA -The four DNA nucleotides -Thermostable Taq polymerase -Buffer -Thermal cycler
A small sample of DNA is needed to act as the template for amplification. Primers target the section of DNA to be replicated. |
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How many cycles does the DNA go through in the thermal cycler? |
30 to 60. |
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What is the first stage in the thermal cycler? |
Denaturation at 95 degrees celsius. Taq polymerase is thermostable and does not denature. |
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What is the second stage in the thermal cycler? |
Cooling to 55 degrees celsius allows the primers to anneal to their complementary bases on the single stranded DNA. |
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What is the third stage in the thermal cycler? |
The DNA is extended by the Taq DNA polymerase in a 5' to 3' at the Taq polymerase's optimum temperature of 72 degrees celsius. |
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What is DNA sequencing? |
The determination of the order of the nucleotide bases in a fragment of DNA. |
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How are dideoxynucleotides used in DNA sequencing? |
They lack a hydroxyl group on carbon 3, and so no more extension of the DNA polymer can occur. Thus, dideoxynucleotides can be known as chain-terminating. The fragment of DNA to be sequenced is added to an appropriate amount of dideoxynucleotides so a full range of fragment lengths are produced. |
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Describe the Sanger sequencing method. |
The primer is radioactively labelled. Four separate PCR tpe incubations are carried out. In each one, a different chain-terminating dideoxynucleotide is used. The different chain terminators produce a different selection of fragment lengths in each of the four incubators. The fragments are in four adjacent lanes in a gel electrophoresis. Once the result has been blotted and exposed to photographic film, the base sequence can be read from the bottom up. |
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What is genetic linkage mapping used for? |
To map the inheritance of many different tpes of genetic marker, such as probe binding sites and restrictive enzyme cut sites. |
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What is a genetic linkage map based on? |
The frequency of recombination between two different markers. |
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What is physical mapping based on? |
The order of genes on each chromosome and the number of base pairs between two markers. |
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What is DNA screening? |
The identification of individuals who are carriers of particular genotypes or markers that are associated with an increased risk of disease. |
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What is gene therapy? |
The replacement of a faulty gene with a new, working gene or the insertion of an extra gene in the hope that the gene product will play a therapeutic role. |
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What is cystic fibrosis? |
A genetic disease caused by the mutant form of the CFTR protein. |
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What is the most common cystic fibrosis causing mutation? |
Deletion of one base triplet, leaving the protein with only 1479 amino acids and not 1480, preventing it from folding properly. |
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What happens due to the mutated gene? |
The mutated gene ensures that the CFTR protein is unable to channel chloride ions and thus cells become more hypertonic. Water is drawn into the cells, making the mucus lining the lungs sticky and impossible for the cilia in the trachea to move. |
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How is cystic fibrosis treated? |
Physiotherapy and drug use to prevent infection. |
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What is Duchenne's muscular dystophy? |
A sex linked recessive condition that causes muscle deterioration during adolescence. |
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How does Duchenne's muscular dystrophy arise? |
Deletion mutations on the gene that is responsible for the synthesis of the body's largest protein, dystrophin, which links the cytoskeleton to the membrane of muscle cells. |
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How are genetic disorders screened for? |
Single locus probes are used to identify common mutations by using probes that hybridise with particular mutant gene sequences. In other cases, normal gene sequences are probed for and any failure of probe hybridisation indicates a deletion has taken place. |
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What is DNA profiling used for? |
To establish beyond reasonable doubt the differences between the genomes of people. It is used in paternity tests, pedigree tests, etc. |
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What are introns? |
Non coding sequences of DNA in between our genes. |
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What are exon gene sequences? |
Sequences that code for proteins. They barely vary from one individual to the next. |
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What are variable number tandem repeats (VNTRs)? |
The most hypervariable DNA found in repeated short sequences of nucleotides. The number of repeats is variable and inherited. |
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How can VNTRs be utilised? |
By examining several simultaneously to identify individuals and confirm family relationships. |
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What are the stages in forensic profiling? |
1) DNA samples are collected and may be amplified using PCR. 2) Individual DNA samples are digested with a restriction enzyme, cutting the DNA at specific sequences around the VNTR sites. Since the VNTR is determined by the number of repeating units, the length of the fragments will differ between individuals. 3) DNA fragments are separated through gel electrophoresis by passing a current through them. DNA has a negative charge and shorter fragments travel farther in the gel. 4) The DNA is denatured into single strands and the fragments blotted onto a membrane. 5) A selection of single locus probes hybridises with complementary sequences on the DNA and can be detected. 6) The position of the probes produces a banding pattern. This is then compared with DNA samples treated in the same way. |
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What is selective breeding? |
Choosing organisms to breed offspring with certain desirable characteristics. |
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What is hybridisation (in agriculture)? |
The crossing of individuals from different varieties in order to produce a predictable phenotype in the F1 generation with dominant characteristics of both breeds. |
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What are transgenics? |
Organisms which contain DNA from two different species. |
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What are cisgenics? |
Organisms which have different genes transferred to them from members of the same species. |
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How can plasmids be genetically modified to produce eukaryotic protein? |
An RNA transcript of the desired gene is harvested from a eukaryotic cell and complementary DNA is manufactured using the enzyme reverse transcriptase. This manufactured gene for the protein is then spliced into a plasmid using ligase. Once the plasmid contains the eukaryotic gene, it is known as a recombinant plasmid. |
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What feature of recombinant plasmids allow them to be selected for culture? |
They usually posses a marker gene, such as for antibiotic resistance. Thus, only these bacteria could be cultured on agar containing that antibiotic. |
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How is bovine somatotrophin (BST) produced? |
The gene is cloned into E.coli and the BST protein produced is purified and administered to cattle by injection. |
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What does BST do? |
Increase milk production as it prevents mammary cell death and increases the duration of lactation. |
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How are transgenic plants engineered? |
By either introducing DNA directly using a particle gun or by using the bacterium Agrobacterium tumefaciens, which contains a plasmid that can integrate genes into a plant cell genome and can therefore be used as a vector in the transfer of DNA from one organism to another. |
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What is the plasmid in agrobacterium called? |
Tumour inducing plasmid (Ti). |
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What does the Ti plasmid do in normal circumstances? |
Causes a tumour or gall to form, which increases the habitat for the bacterium. |
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What must be done to prepare the Ti plasmid for genetic transformation? |
The disease causing gene is disabled. An additional ori sequence is added to allow the plasmid to be grown in a lab in E.coli. Marker genes are added. The desired gene for transfer is also added. |
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How is the desired gene for transfer added? |
The sequence is identified and synthesised or removed from its source using a restriction enzyme. The plasmid must then be cut with the same restriction enzyme and ligase used to seal the gene into the plasmid. |
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How can the plant cells be transfected? |
Through either applying the recombinant plasmid agrobacterium directly to wounded plant tissue or incubating it with plant protoplasts in the laboratory. |
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How are protoplasts used? |
They are prepared by digesting the cell walls with cellulase. Then they are incubated with the Agrobacterium in a selective medium. This allows only the plant cells which have taken up the recombinant plasmid to grow. |
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What are meristems? |
The growing points of plants. They are to totipotent, meaning they can differentiate into any different cell type. |
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How do cells become differentiated? |
Each cell type has a different combination of genes being expressed, with some switched on and some switched off. This effect is permanent, and explains why specialised cells can no longer differentiate into different types. |
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What does pluripotent mean? |
Cells such as adult stem cells which can only produce a restricted range of cell types. |
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What is significant about embryonic stem cells? |
They can differentiate into any fell type, but this ability is lost as the organism develops. |
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How can gene expression be controlled in eukaryotes? |
Changes to the histone protein of the nucleosome.
Methylation of cytosine nucleotides. |
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How can gene switching in prokaryotes be described? Use the lac operon mechanism as an example. |
The system has: -a regulator gene which codes for a repressor molecule. -the repressor molecule which binds to the operator or the inducer. -a structural gene that makes beta galactosidase, an enzyme which breaks down lactose. -an operator consisting of a length of DNA upstream of the structural gene. -an inducer molecule (lactose) which binds to the repressor molecule.
When no lactose is present, the repressor gene makes the repressor molecule. This attached to the operator so that the transcription enzyme cannot read the structural gene. No beta galactosidase is made and the bacterium does not waste energy making an enzyme it doesn't need.
When lactose is present, it acts as the inducer to cause the production of beta gal |
