There is practical work within units (1) - (3), while unit (d) is based on research and experimentation entirely.
UNIT 1: Cell and Molecular Biology - The structure & function of prokaryotic and eukaryotic cells - The structure and function of cell components - Molecular interaction in cell events - Applications of DNA technology
UNIT 2: Environmental Biology - Circulation in ecosystems - Interaction in ecosystems - Human impact on the environment
HALF UNIT 3: Physiology, Health and Exercise - Exercise and the cardiovascular system - Exercise and metabolism
HALF UNIT 4: Investigation - A scientific investigation into a syllabus-related topic that is designed, carried out and written up as a 2000-2500 word report, worth 20% of the final marks.
Methods of Learning
Powerpoint presentations, directed reading and laboratory work
Practical work emphasises experimental skills used to investigate basic issues associated with each topic
You are also expected to devote considerable effort on the planning, evaluation and writing up of your investigations
Homework is set on a regular weekly basis. At least 3 hours per week in self study at home!
- Short essay type answers like those in sections C and D of the external examination
- Data Handling questions like those in sections B, C and D of the external examination and unit tests
- Writing up learning outcomes to produce summaries
- Revision for unit assessments, topic tests etc .
- Planning, carrying out and writing up the investigation
- Writing up class practical activities
End of unit tests Unit awards are obtained by taking a NAB at level C only. 65% is required to pass the test
Experimental reports You will carry out, record and write up, in the form of a report, one experiment. To achieve a course award in the final examination, all three unit tests and the experimental report must be passed
End of topic tests A/B tests are taken at the end of every unit and prelims are held during the year also
External Assessment 20 marks 4 questions Short answer Unit 3 only SECTION C 55 marks 7 questions Short/Extended/data qu’s Units 1 and 2 only SECTION B 25 marks 25 Multiple choice Units 1 and 2 only SECTION A 2 1/2 hour external examination with a total of 100 marks (worth 80%)
A good Advanced Higher pass will enhance an application for courses at College or University level, especially of Biological subjects … any course really (it shows that you have an excellent brain!)
In terms of University admission, Advanced Higher Biology is rated equivalent to A Level Biology, but the investigative research in Advanced Higher provides a very valuable opportunity at this level of study
Advanced Higher Biology UNIT 1 Cell & Molecular Biology
STRUCTURE, FUNCTION & GROWTH OF PROKARYOTIC & EUKARYOTIC CELLS
Prokaryotic and Eukaryotic Cells
All living creatures are made up of CELLS , small membrane bound units filled with aqueous solutions of chemicals, which have the ability to create copies of themselves by growing and dividing.
[The sizes of cells and organelles]
Living organisms can be classified into 3 major domains:
Prokaryotes and Eukaryotes are 2 distinct cell types with STRUCTURAL differences
The Prokaryotic Cell
Simply stated, prokaryotes are molecules surrounded by a membrane and cell wall.
Lack a membrane bound nucleus enclosing the DNA
DNA is present as a single circular molecule called a BACTERIAL CHROMOSOME
DNA is naked having no associated histone proteins
No membrane bound organelles
Apart from the DNA nucleoid, there is little internal structure apart from dissolved substances and a large number of RIBOSOMES essential for PROTEIN SYNTHESIS
The cytosol is an effective site for bacterial cell metabolism. This allows bacteria to adapt quickly to changing nutritional conditions, but means the regulation of genetic and metabolic activity has to be tightly regulated.
Divide by BINARY FISSION
Some prokaryotic cells have external whip-like FLAGELLA for locomotion or hair like PILI for adhesion.
Prokaryotic cells come in multiple shapes: cocci (round), baccilli (rods), and spirilla or spirochetes (helical cells).
External Prokaryotic Structures
Contains PEPTIDOGLYCAN (only found in bacteria). Large complex molecule consisting of polysaccharide polymers cross-linked by short chains of amino acids
Sometimes the cell wall is further surrounded by a gelatinous polysaccharide sheath called an attach CAPSULE , GLYCOCALYX or SLIME LAYER
Basic structure of the phospholipid bilayer is the same for all bacteria
Flagella Motile bacteria usually have long, thin appendages called FLAGELLA . These protein sub-units are used to propel bacteria through liquids
Pili or Fimbrae
A pilus (Latin; plural : pili ) is a hairlike protein structure on the surface of a bacterial cell, required for bacterial conjugation (transfer of genetic material)
A fimbrium (Latin; plural: fimbria ) is a short pilus that is used to attach the cell to a surface. Mutant bacteria that lack fimbria cannot adhere to their usual target surfaces and, thus, cannot cause diseases.
Spores & Cysts
These are produced by some bacteria to survive unfavourable environmental conditions. Dormant forms are metabolically inactive and only germinate under suitable conditions
ENDOSPORES : a dormant, tough, non-reproductive structure produced by a small number of bacteria. The primary function of most endospores is to ensure the survival of a bacterium through periods of environmental stress. They are therefore resistant to ultraviolet and gamma radiation , desiccation , lysozyme , temperature , starvation , and chemical disinfectants . Endospores are commonly found in soil and water, where they may survive for long periods of time e.g. Clostridium (tetanus, gas gangrene), Bacillus (anthrax) CYSTS : also dormant, but unlike endospores are not resistant to heating at high temperatures
Main method is using the GRAM’S STAIN
This separates bacteria into GRAM-POSITIVE (purple) and GRAM-NEGATIVE (red) depending on the percentage of PEPTIDOGLYCAN in the cell walls
- GRAM-POSITIVE bacteria have a cell wall only 1 layer thick
- GRAM-NEGATIVE bacteria have a cell wall several layers thick
More complex multicellular organisms e.g. plants, animals, fungi and also many single-celled organisms e.g. amoeba, yeast
Possess an NUCLEUS and other organelles all of which are surrounded by a MEMBRANE , which divided the cell up into compartments
COMPARTMENTALISATION: very important !
Molecules are ‘concentrated’ together, increases rate of reactions
Keeps reactive molecules away from other parts of the cell that may be affected by them
Large work surface area … many enzymes are bound in membranes
The basic eukaryotic cell contains the following:
- membrane-bound nucleus
- plasma membrane
- glycocalyx (components external to the plasma
- cytoplasm (semifluid)
- cytoskeleton – microfilaments, intermediate filaments and
microtubules that suspend organelles, give
shape, and allow motion
- presence of characteristic membrane
enclosed subcellular organelles e.g.
mitochondria, golgi, rER, sER etc
Plant & Animal Cells
For ANIMAL CELLS only:
Peroxisomes & Lysosomes often present
Some have microvilli on their surface
Centrioles organise spindle fibres during cell division
For PLANT CELLS only:
Cell walls made from cellulose
Communication with neighbouring cells occurs through plasmodesmata
Usually a large central vacuole
Photosynthesis occurs in cells containing chloroplasts
[Stick in & label plant & animal cell diags]
A lipid/protein/carbohydrate complex, providing a barrier and containing transport and signalling systems.
Double membrane surrounding the chromosomes and the nucleolus. Pores allow specific communication with the cytoplasm. The nucleolus is a site for synthesis of RNA making up the ribosome
Surrounded by a double membrane with a series of folds called cristae.
Functions in energy production through metabolism.
Contains its own DNA, and is believed to have originated as a captured bacterium.
Rough endoplasmic reticulum (RER)
Rough endoplasmic reticulum (RER)
A network of interconnected membranes forming channels within the cell.
Covered with ribosomes (causing the "rough" appearance) which are in the process of synthesizing proteins for secretion or localization in membranes.
Protein and RNA complex responsible for protein synthesis
A series of stacked membranes. Vesicles (small membrane surrounded bags) carry materials from the RER to the Golgi apparatus.
Vesicles move between the stacks while the proteins are "processed" to a mature form.
Vesicles then carry newly formed membrane and secreted proteins to their final destinations including secretion or membrane localisation.
Centrioles are found only in animal cells. They function in cell division.
A membrane bound organelle that is responsible for degrading proteins and membranes in the cell, and also helps degrade materials ingested by the cell.
Peroxisomes or Microbodies
Produce and degrade hydrogen peroxide, a toxic compound that can be produced during metabolism
Surrounded by a double membrane, containing stacked thylakoid membranes.
Responsible for photosynthesis, the trapping of light energy for the synthesis of sugars.
Contains DNA, and like mitochondria is believed to have originated as a captured bacterium.
Membrane surrounded "bags" that contain water and storage materials in plants.
Plants have a rigid cell wall in addition to their cell membranes. They provide support for the plant.
Similarities between P & E cells
Prokaryotes & Eukaryotes are CHEMICALLY & METABOLICALLY similar:
Both have genetic material
Both have a cell membrane
Both have a cytosol
Both have ribosomes
Both contain nucleic acids, proteins, carbohydrates & lipids
Both use similar reactions for storing energy and metabolic activities e.g. building proteins
Differences between P & E cells
Main differences are STRUCTURAL :
Cell size ranges from 10 – 150um Cell size ranges from 0.5um to 100um No mucilaginous capsule present (numerous internal structures present including microtubules, ER, Golgi, secretory vesicles etc) Mucilaginous capsule Have cilia or flagella (for movement) Have pili & fimbriae (for adhesion) and flagella (for propulsion) Membrane bound organelles (compartmentalisation) No membrane bound organelles Cell walls, if present, made of cellulose (chitin in fungi) Cell walls made of peptidoglycan (Thickness of wall depends on whether the cell is Gram +ve or –ve) Membrane bound nucleus No membrane bound nucleus EUKARYOTES PROKARYOTES
Comparison of Prokaryotic and Eukaryotic Cells
Cell Growth & The Cell Cycle
Living things can be distinguished from non-living things by their ability to REPRODUCE
This characteristic is based on cells being ability to DIVIDE
What is DNA and where is it stored?
The nucleus is a membrane bound organelle that contains the genetic information in the form of chromatin, highly folded ribbon-like complexes of deoxyribonucleic acid (DNA) and a class of proteins called histones .
Cell division allows organisms to grow, develop, to rweplace dead cells and to repair tissue
This is a CONTINUAL PROCESS
The length of the cell cycle depends on the type of cell and external factors e.g. temp, O2 supply etc
Bacterial cells – 20 mins
Liver cells divide only once a year or only if the need arises e.g. injury
Skin cells – all the time
Nerve & muscle cells don’t divide at all in a mature adult
The Cell Cycle
Stages in the Cell Cycle:
Cell Growth and The Cell Cycle
A eukaryotic cell cannot divide into two, the two into four, etc. unless two processes alternate:
doubling of its genome ( DNA ) in S phase (synthesis phase) of the cell cycle;
halving of that genome during mitosis ( M phase )
The period between M and S is called G 1 ; that between S and M is G 2 .
For a new cell to be produced …
The quantity of DNA must double – DNA replication
Must be copied EXACTLY
Due to the brief flurry of cytological activity during cell division, the cycle is divided up into 2 parts:
INTERPHASE (G1, S, G2 phases)
MITOTIC PHASE (M phase)
So, the cell cycle consists of:
G 1 = growth and preparation of the chromosomes for replication
S = synthesis of DNA (and centrosomes )
G 2 = preparation for
M = mitosis
When a cell is in any phase of the cell cycle other than mitosis, it is often said to be in Interphase .
What is (and is not) mitosis?
Mitosis is nuclear division plus cytokinesis , and produces two identical daughter cells during prophase, metaphase, anaphase, and telophase.
Interphase is often included in discussions of mitosis, but interphase is technically not part of mitosis, but rather encompasses stages G1 , S , and G2 of the cell cycle .
The cell is engaged in metabolic activity and performing its preparation for mitosis (the next four phases that lead up to and include nuclear division).
Chromosomes are not clearly discerned in the nucleus, although a dark spot called the nucleolus may be visible.
The cell may contain a pair of centrioles (or microtubule organising centres in plants) both of which are organisational sites for microtubules.
Chromatin in the nucleus begins to condense and becomes visible in the light microscope as chromosomes.
The nucleolus disappears. Centrioles begin moving to opposite ends of the cell and fibres extend from the centromeres.
Some fibres cross the cell to form the mitotic spindle.
The nuclear membrane dissolves, marking the beginning of metaphase.
Proteins attach to the centromeres creating the kinetochores . Microtubules attach at the kinetochores and the chromosomes begin moving.
Spindle fibres align the chromosomes along the middle of the cell nucleus. This line is referred to as the metaphase plate.
This organisation helps to ensure that in the next phase, when the chromosomes are separated, each new nucleus will receive one copy of each chromosome.
The paired chromosomes separate at the kinetochores and move to opposite sides of the cell.
Motion results from a combination of kinetochore movement along the spindle microtubules and through the physical interaction of polar microtubules.
Chromatids arrive at opposite poles of cell, and new membranes form around the daughter nuclei.
The chromosomes disperse and are no longer visible under the light microscope.
The spindle fibres disperse, and cytokinesis or the splitting of the cell may also begin during this stage.
In animal cells, cytokinesis results when a fibre ring composed of a protein called actin around the centre of the cell contracts pinching the cell into two daughter cells , each with one nucleus.
In plant cells, the rigid wall requires that a cell plate be synthesised between the two daughter cells.
C yto k inesis
C alvin K lein
Before a cell can divide, it must duplicate all its DNA. In eukaryotes, this occurs during S phase of the cell cycle .
Recap the steps in DNA replication ….
A portion of the double helix is unwound by a helicase .
A molecule of a DNA polymerase binds to one strand of the DNA and begins moving along it in the 3' to 5' direction, using it as a template for assembling a leading strand of nucleotides and reforming a double helix.
Because DNA synthesis can only occur 5' to 3', a molecule of a second type of DNA polymerase binds to the other template strand as the double helix opens. This molecule must synthesize discontinuous segments of polynucleotides (called Okazaki fragments). Another enzyme, DNA ligase I then stitches these together into the lagging strand .
DNA Replication is Semiconservative
When the replication process is complete, two DNA molecules — identical to each other and identical to the original — have been produced. Each strand of the original molecule has remained intact as it served as the template for the synthesis of a complementary strand.
This mode of replication is described as semi-conservative : one-half of each new molecule of DNA is old ; one-half new .
Interphase: G1, S and G2 phases
Lasts much longer than the M phase
Sometimes referred to as the ‘resting’ phase – this is UNTRUE as although it doesn’t look like much is happening, in biochemical terms, this is a very active period of CELL GROWTH & METABOLISM
Protein synthesis takes place
Cytoplasmic organelles are synthesised
The cell grows and replicates its chromosomes [only during S phase]
Interphase is divided into 3 parts:
(1) G1 – First ‘Gap’ phase ( During this time the cell is very active, growing and carrying out metabolic processes )
(2) S - DNA replication ( The 'S' stands for synthesis as during this phase DNA is synthesized in the process of replication. Each chromosome becomes two sister chromatids)
(3) G2 - Second ‘Gap’ phase ( In this period mitochondria and other organelles are divided so that each daughter cell will have an equal number of organelles)
Mitosis: the M phase
Interphase is followed by M Phase which consists of mitosis and cytokinesis.
Mitosis is the division of the contents of the nucleus (PMAT), whilst cytokinesis (CK) refers to the division of the cytoplasm.
Cell division involves mitosis and cytokinesis. The growth of an organism and the replacement of its cells for tissue repair both depend on mitosis and cytokinesis.
Control of the Cell Cycle
A central mechanism is used to assess the status of the cell as it progresses through the cycle. This system works through 3 main checkpoints:
G1 Checkpoint: towards the end of the S phase.Size of the cell is assessed - if sufficient growth has occurred i.e. cell large enough for division, then S phase can proceed
G2 Checkpoint: the success of DNA replication is monitored. If successful the cell cycle will continue to mitosis
M Checkpoint: during metaphase prior to anaphase and telophase triggers exit from from mitosis and cytokinesis and entry into next G1 phase for daughter cells
Abnormal Cell Division : Cancer cells
Normal cell development will break down if the control of cell division , cell growth or cell death fails
If cell division or cell growth fails, TUMOURS arise
These can either be benign - don’t cause serious problems and can be removed by surgery or malignant - enter the circulation, migrate and proliferate to form new tumours in new areas of the body. This is called METASTASIS
Checkpoints: Where stop and start signals regulate the cycle; register internal and external cell signals which report the state of crucial processes and if the cycle should proceed.
Chemotherapy is the use of anti-cancer (cytotoxic) drugs to destroy cancer cells (including leukaemias and lymphomas). There are over 50 different chemotherapy drugs and some are given on their own, but often several drugs may be combined (this is known as combination chemotherapy ).
Chemotherapy may be used alone to treat some types of cancer. Sometimes it can be used together with other types of treatment such as surgery, radiotherapy, hormonal therapy, immunotherapy, or a combination of these.
How do chemotherapy drugs work?
Chemotherapy drugs interfere with the ability of a cancer cell to divide and reproduce itself. As the drugs are carried in the blood, they can reach cancer cells all over the body. The chemotherapy drugs are taken up by dividing cells, including some normal cells such as those in the lining of the mouth, the bone marrow (which makes blood cells), the hair follicles, and the digestive system. Healthy cells can repair the damage caused by chemotherapy but cancer cells cannot and so they eventually die.
Chemotherapy drugs damage cancer cells in different ways. If a combination of drugs is used, each drug is chosen because of its different effects. Unfortunately, as the chemotherapy drugs can also affect some of the normal cells in your body, they can cause unpleasant side effects. However, damage to the normal cells is usually temporary and most side effects will disappear once the treatment is over.
Chemotherapy is carefully planned so that it destroys more and more of the cancer cells during the course of treatment, but does not destroy the normal cells and tissues.
Multicellular organisms are created from a complex organization of cooperating cells.
Some cells provide protection; some give structural support or assist in locomotion; others offer a means of transporting nutrients.
All cells develop and function as part of the organized system -- the organism -- they make up. There must be new mechanisms for cell to cell communication and regulation.
In humans, there are 10 14 cells comprising 200 kinds of tissues!
Each of us originated as a single, simple-looking cell -- a fertilized egg, or zygote -- so tiny that it can barely be seen without a microscope. ( A human egg cell is about 1/100th of a centimetre in diameter, or a bit smaller than the width of a human hair .)
Shortly after fertilization, the zygote begins dividing, replicating itself again and again. Before long, a growing mass, or blastula, of dozens, then hundreds, then thousands of cells called stem cells forms; each stem cell is only one-fourth to one-tenth the diameter of the original zygote, but otherwise nearly identical to it
Every nucleus of every cell has the same set of genes. A heart cell nucleus contains skin cell genes, as well as the genes that instruct stomach cells how to absorb nutrients.
Therefore, for cells to differentiate, certain genes must somehow be activated, while others remain inactive.
Genes instruct each cell how and when to build the proteins that allow it to create the structures, and ultimately perform the functions, specific to its type of cell.
Gene Regulation in Bacteria
Bacteria adapt to changes in their surroundings by using regulatory proteins to turn groups of genes on and off in response to various environmental signals
The DNA of Escherichia coli is sufficient to encode about 4000 proteins, but only a fraction of these are made at any one time. E. coli regulates the expression of many of its genes according to the food sources that are available to it.
The lac operon
The best understood cell system for explaining control through genetic induction is the lac operon
Jacob & Monod (1961) - regulation of lactose
metabolism in E.coli
Composed of 3 segments, or loci of DNA:
1. The REGULATOR - composed of gene that codes for a repressor protein which can repress the operon.
2. The CONTROL locus - consists of a promotor and the operator - can start transcription of the structural genes
3. The STRUCTURAL locus - contains structural genes encoding the enzyme -galactosidase
The lac operon In an E. Coli cell growing in the absence of lactose , a repressor protein binds to the operator, preventing RNA polymerase from transcribing the lac operon's genes. The operon is OFF When the inducer, lactose , is added, it binds to the repressor and changes the repressor's shape so as to eliminate binding to the operator. As long as the operator remains free of the repressor, RNA polymerase that recognizes the promoter can transcribe the operon's structural genes into mRNA. The operon is ON
The lac operon Lactose absent :
The lac operon Lactose present :
Mammalian Cell Culture
The ability to grow cells in culture i.e. in the lab, is essential for biotechnology and research
Applications of cell culture 1
RESEARCH (small scale usage)
growing bacterial cells for basic gene manipulation
culturing mammalian cells to observe the effects of drugs and hormones on the functioning of cells e.g. cancer studies
producing new plants
Applications of cell culture 2
BIOTECHNOLOGY (large scale usage)
agriculture e.g. silage production
pharmaceuticals e.g. genetically engineered bacteria to produce insulin
food production e.g. brewing and baking
biodegradation e.g. sewage treatment
Conditions needed for cell culture
In order for cells to grow, the conditions must be
just right for each cell type. The cytologist must
therefore consider the following:
Type of growth container or fermenter
Method for monitoring cell growth
Safety measures and implications
To avoid contamination of growth media and cultures
All inanimate and living objects, including the atmosphere carry large numbers of microorganisms.
A variety of techniques can be used to provide these conditions:
e.g. sterilisation of all utensils and media using heat . For example, using an autoclave (steam under pressure, necessary for bacterial spores) Growth of pure cultures
They are everywhere!
They highly adaptable to their surrounding environment
They are relatively easy to culture
They incredibly diverse and are able to colonise very extreme conditions e.g. salt pans, hydrothermal vents in the ocean floor
Classes of Microorganisms
There are 2 recognised categories of micro-organism:
1. Unicellular Algae / PHOTOTROPHS : use sunlight to make their own food 1. Bacteria & Yeasts (Fungi) / HETEROTROPHS : need more complex media containing an organic carbon source and other compounds e.g. amino acids
Culture & Uses
Food industry - cheese production, baking, wine & beer
Chemical production e.g. acetone
Bases of food chains
Commensal bacteria in digestive tract
Production of therapeutic compounds e.g. insulin
[See Scholar: Batch & Continuous culture]
Microbial Growth Culture Requirements
A few litres can be made in the lab
Thousands of litres can be made industrially
Micro-organisms are grown in a medium that supplies them with all nutrients necessary for growth.
This depends on …
the type of cell
the final purpose of the cell
Microbial Growth Culture Requirements
Important factors that must always be considered are:
the nutrient media
Unicellular algae , bacteria & yeast can be grown as batch cultures - no dilution is needed until max. density is reached. Growth can be limited by nutrient availability i.e. at the end of exponential growth
Chosen to imitate an organism’s natural environment
Generally supplies all the essential nutrients
A medium is classed as any solid or liquid preparation specifically for growth, storage or transport of micro-organisms
Must be at the correct pH and the correct gaseous concentration for the organisms to grow
There are 2 types of media commonly used:
1. Complex media - this has one or more crude sources of nutrients and their exact chemical composition and components are not known. Generally used for routine cultures 2. Defined media - otherwise known as synthetic media containing chemically known compounds and components which are in a relatively pure form REMEMBER: all media must be STERILE before use !!!
Mammalian Cell Culture
Many animal cells and tissues can be removed from an organism and cultured artifically. This allows the cell’s activities to be investigated e.g. control of the cell cycle
The process of culturing Mammalian Cells
Once the cells are obtained from animal tissues or other cell lines they are placed in a flat culture vessel that lies on its side
The cells stick or adhere to the inside of the vessel as they grow in the medium Most animal cells are ‘ANCHORAGE-DEPENDENT’ i.e. they need something to hold on to These cells usually form a monolayer that will eventually cover the entire surface of the medium
At this point, called confluence , it is necessary to subculture the cells into a fresh medium N.B. Cells that are associated with body fluids such as blood cells are NON-ANCHORAGE DEPENDENT and can be grown in suspension. Again, it is necessary to regularly subculture the cells into fresh medium N.B. All media and culture vessels are STERILISED to prevent the growth of micro-organisms
Mammalian Cell Growth Media
- mixture of glucose , amino acids , salts , water
- sometimes BASIC GROWTH SERUM is added
[This is animal serum prepared from blood and contains additional factors e.g. Platelet Derived Growth Factor,which enhances growth, 5-10% added or Fetal Bovine Serum (FBS) ]
- pH indicator e.g. phenol red: this shows
changes in pH due to waste production
(pH decreases red to yellow )
* Finally, the media must be incubated at the appropriate temperature for the chosen cells e.g. human cells - 37 o c *
Categories Of Mammalian Cell Cultures
There are 2 categories of animal cell cultures:
(1) Primary cultures:
These cells are taken directly from fresh tissue.
The disadvantage is that the cells have a limited lifespan; the cells only divide so many times in culture, so therefore long term culturing is difficult
Process of Cell Collection
The cells are treated with a proteolytic enzyme e.g. trypsin, to separate out the fragments into single cells.
The advantage of this process is that cells can be collected and cloned .
This is useful to isolate a mutant cell line i.e. deriving secondary cell cultures otherwise known as ...
(2) Continuous Cell Lines
These cells have an acquired capacity for infinite growth and division [they are immortal]
They are derived from tumours or the cells have been transplanted [neo-plastic - produce cancer if transplanted into animals] so they have lost their sensitivity to factors associated with growth control.
Generally, these cells will lose their anchorage dependence facility and so are often easier to culture
Continuous Cell Lines
The advantage of using continuous cell lines is that they can be cloned .
This allows easy :
isolation of mutant cells
investigation of cell growth
production of hybrid cells in biotechnology
This routine procedure is used to produce important pharmaceuticals e.g. vaccines and hormones
Bacterial & Fungal Cultures
Much easier to grow than mammalian cells !
Bacteria and Fungi require much simpler growth media requirements and culture conditions compared to animal cells.
[See previous notes]
Plant Tissue Culture
One major problem in plant breeding is that crosses can only be made between closely related parental types. This makes it very difficult to introduce new genes into a plant species.
The solution to this problem is PROTOPLAST FUSION - protoplasts of different plants are mixed and fused together. These form a binucleate cell containing a nucleus from both parental types
Protoplast = actively metabolising part of cell minus cell wall [cell wall digested by enzymes]
Process of Plant Cell Culture
1. Plant cells treated with cellulase & pectinase to remove the cell wall which is composed of cellulose, pectin and small amount of hemicellulose. These enzymes only break down the cell wall leaving the plasma membrane intact
2. The cells are then incubated with a mineral salt solution containing mannitol for several hours. This sugar exerts osmotic pressure causing PLASMOLYSIS leading to easier digestion 3. In order for the protoplasts to grow they must be put in a suitable medium to encourage cell wall growth
4. EXPLANTS (small pieces of young growing plant tissue e.g. root, shoot, bud or leaf) can be taken and grown in a suitable medium containing plant growth regulators (growth hormones e.g. auxins and cytokinins whuch cause tissue differentiation). Cell proliferation produces a CALLUS (a mass of dividing, undifferentiated cells) 5. With continued sub-culturing and changing the balance of growth regulators, the new roots and shoots can be planted out to regenerate a complete plant !
All plant cells are totipotent - they each have the ability to express the full genetic potential of that plant
STRUCTURE & FUNCTION OF CELL COMPONENTS
Living systems are composed of a limited number of elements namely…
The carbon atom is of central biological importance as it can form 4 covalent bonds with other atoms
This allows a variety of complex molecules to be constructed
Many functional chemical groups are also associated with biological molecules as they are important in biological systems
Many biologically important molecules are polymers composed of monomers linked together
Two monomers are joined together by removing water molecules. This is called a CONDENSATION reaction or DEHYDRATION synthesis
This can be reversed by adding (back) water -> HYDROLYSIS
This is an important feature of cell metabolism
Making and breaking chemical bonds involves ENERGY
Synthesising more complex structures REQUIRES energy. These are called ANABOLIC or BIOSYNTHETIC reactions
If there is little overall change in energy, the reactions are reversible
Cell metabolism is tightly controlled to avoid energy chaos
Composed of CARBON, HYDROGEN & OXYGEN
MONOSACCHARIDES ‘Single Sugars’ e.g. glucose, fructose - General formula (CH 2 O)n - classified by number of carbons they have n = 3 TRIOSE n = 5 PENTOSE n = 6 HEXOSE - structure can vary greatly depending on the number of C atoms and the arrangement of H and O atoms
Glucose (C 6 H 12 O 6 )
Can exist in different forms depending on the position of the carbonyl group (C=O) on the terminal carbon
Variations of C 6 H 12 O 6 are called isomers
If OH group on C5 projects to the right = D Form (most common)
on left = L Form
D-GLUCOSE = straight chain form of glucose (C 6 H 12 O 6 )
In solution, glucose adopts a cyclic form where C 1 and C 5 are linked by an oxygen atom giving a ring structure ( see diagram )
Depending on the position of the -OH group on C 1 whether:
( ) alpha - below C 1
( ) beta - above C 1
In solution the equilibrium proportions of the three forms are approximately 38% to 62% to 0.02% straight chain glucose at any given time
The Glycosidic Bond
2 monomers (monosaccharides) can be linked by DEHYDRATION SYNTHESIS or the CONDENSATION REACTION , to give a disaccharide
The carbohydrate’s name is defined by the component monomers and the way the bond is arranged
Common disaccharides are :
SUCROSE = Glucose + Fructose
LACTOSE = Glucose + Galactose
Long chains of simple sugars e.g. starch , glycogen and cellulose
If the repeating monomers are the same , they form a homopolymer . If they are different they form a heteropolymer
Polysaccharides are insoluble in water and so make ideal storage compounds
The following three polysaccharides are all homopolymers of glucose but they have different functions and properties depending on their structure
Found in plants
Helical arrangement of glucose
Storage polysaccharide of energy
Can be easily hydrolysed to release monomers of glucose for energy
Starch test : turns iodine from dark brown to blue/black
Storage compound in animals, generally found in the liver
Polymer of glucose linked by 1-6 bonds and 1-4 bonds
Short term energy store
Plays a role in homeostatic control of blood sugar level
Remains dark brown with iodine
Storage compound in plants
Parallel chain arrangement linked by 1-4 glycosidic bonds and hydrogen bonding between parallel chains
Doesn’t stain with iodine
Very tough arrangement of fibres due to structural arrangement
most abundant organic material on Earth
Most animals lack cellulase, the enzyme needed to breakdown the component monomers
A homopolysaccharide similar to cellulose in structure. Component of many insect exoskeletons - very strong and rigid; also resistant to chemicals.
A heteropolymer found in skin and connective tissue of vertebrates
Summary of Carbohydrate Functions
Immediate respiratory substrate e.g. glucose
Energy stores e.g. glycogen in mammals, starch in plants
Structural components e.g. cellulose in plant cell walls, chitin in insect exoskeleton, pentose sugars (ribose & deoxyribose in RNA & DNA)
Metabolites i.e. intermediates in biochemical pathways
Cell to cell attachment molecules e.g. glycoproteins or glycolipids on the plasma membrane
Transport e.g. sucrose in plant phloem tissue
Structure & Function of Lipids
Lipids are organic compounds found in every type of plant and animal cell.
They contain the elements CARBON, HYDROGEN and OXYGEN [ but less O 2 than in carbohydrates ]
All lipids are INSOLUBLE in WATER
Lipids have many important functions:
In cell membrane structure - Mechanical Protection
Hormones - Electrical Insulation of Nerves
Energy storage molecules - Waterproofing & Buoyancy
FATS: Solid at room temperature
SATURATED FATS : all available bonds are occupied by Hydrogen
Most animal fats are saturated e.g. butter, lard
OILS: Liquid at room temperature
UNSATURATED FATS: contain C-C double bonds in the molecule therefore kinks are introduced.
Oils tend to be more available in plants e.g. sunflower oil, olive oil
Type of Lipids
3 types of lipids which are important to cells:
Most common type of lipid
3 fatty acids and a glycerol molecule are linked by an ester bond formed during dehydration synthesis
Same as triglycerides except one of the fatty acids molecules is replaced by a phosphate group (PO 4 3 -)
The phosphate group is polar and so is attracted to water – therefore the phospholipid has two distinct ‘ends’
A hydrophilic end (‘water loving’) that dissolves in water and a hydrophobic end (‘water hating’) that is repelled by water
Very different structure – 4 carbon rings with variety of different side chains
The properties of triglycerides are determined by their constituent fatty acids
DEHYDRATION SYNTHESIS occurs between the hydroxyl group of the glycerol molecule and the carboxyl groups of the fatty acid molecule producing an ester
Main function = ENERGY STORE e.g. camel hump
The form in which fatty acids are transported round the body and stored is adipose tissue
Similar to triglycerides but one fatty acid is replaced by a phosphate group which often has other groups attached
Usually one fatty acid is saturated and one is unsaturated. Most common phospholipid in animal tissue is PHOSPHATIDYLCHOLINE
The phospholipid has two distinctive ends:
HYDROPHILIC HEAD that dissolves in water
HYDROPHOBIC TAIL that repels water
This property causes phospholipids to spontaneously form bilayers
Functions of Phospholipids
Essential components of cells and organelle membranes
Components of lung surfactants
STRUCTURE & FUNCTIONS OF PROTEINS
Proteins are essential in biological systems as controls e.g enzymes and structural elements e.g. cytoskeleton
Proteins are heteropolymers as they are made up of different amino acids (20 different types)
The type and order of amino acids determines the structure and function of proteins allowing them to carry out many different roles
Amino acids are characterised by the amino group (NH 2 ) and the carboxylic acid (COOH)
These are attached to a central carbon atom which also carries a hydrogen
The side chains are variable, the ‘R’ group can be joined here
At neutral pH, amino acids exist in ionised forms. Once joined, the charges on amino acids disappear.
This gives the amino acid it’s unique chemical properties and specific shape.
The R group can be classified as acidic, basic, uncharged polar or non-polar
Proteins are made by joining amino acids together by an amide linkage / peptide bond
A chain of amino acids is called a polypeptide
The peptide bond is formed by DEHYDRATION SYNTHESIS or a condensation reaction between the carboxyl group of one amino acid and the amine group of the next amino acid
Amino acids joined in this way are called residues
The Peptide Bond
The Peptide bond is very strong
C-N bond is planar (flat) so peptide bond allows NO rotation
The single bonds either side DO allow rotation of the residues, so polypeptide chains are flexible
Chemical bonding is critical in determining a protein’s shape and the different types of bonds are important for different levels of protein structure
PEPTIDE BOND = COVALENT BOND = VERY STRONG
In higher order protein structures, weaker interactions are important too.These include:
Van der Waals interactions
Hydrophobic interactions between R groups
Primary Structure (1 o )
Primary structure refers to the "linear" sequence of amino acids.
The amino end or N terminus is positioned to the left. The carboxyl end or C terminus is positioned to the right
N C Generally 3 or 1 letter abbreviations are used to denote amino acids when primary structures are drawn
Secondary Structure (2 o )
Secondary structure is "local" ordered structure brought about via hydrogen bonding mainly within the peptide backbone
A single polypeptide many contain several secondary structures
The most common secondary structure elements in proteins are the alpha ( ) helix and the beta ( ) sheet (sometime called b pleated sheet)
Tertiary Structure (3 o )
This describes the way in which the polypeptide folds to give the final structure of the protein.
The 3 o structure is determined by hydrophobic interactions which place the amino acids non-polar R groups towards the centre of the molecule
In many proteins an additional important type of bond is the disulphide bond. This bond forms between sulphydryl (SH) groups on cysteine residues; so may be formed between 2 different polypeptides or within the polypeptide itself.
Within any tertiary structure, parts of the amino acid sequence may adopt an -helix, -sheet or more complex sheet arrangements e.g. myoglobin
The ion group is a prosthetic group – a non-protein group associated with a folded protein
If the attached group is :
CARBOHYDRATE = Glycoprotein
LIPID = Lipoprotein
NUCLEIC ACID = Nucleoprotein
These are known as conjugated proteins
As proteins have a relatively stable structure in a cellular environment, it is remarkable that the forces that hold them together can be easily disrupted if the chemical environment changes or the sequence of amino acids is changed
The polypeptide chain is coiled into a right handed helix by Hydrogen bonding (stabilises the helix) between the NH group of the peptide and the C=O of the peptide bond, four residues away from it
The polypeptide chains are linked together in a side by side configuration by hydrogen bonding. Beta sheets can be either parallel or anti-parallel depending on the orientation of the constituent parts
Quaternary Structure (4 o )
Proteins that are composed of 2 or more polypeptide sub-units
Nucleic Acids [revise Higher notes]
DNA and RNA are information carrying molecules
DNA : info storage & transmission
RNA : protein synthesis
Simple chemical structure based on a SUGAR PHOSPHATE BACKBONE
Coding part made of 4 nitrogenous bases which arrange themselves in pairs
This enables a massive variety and diversity of proteins to be built
[Diagram of Nas/Nucleotides]
Monomer of nucleic acid
Consists of 3 main parts :
a PENTOSE sugar (deoxyribose/ribose)
a PHOSPHATE group (PO 4 2- )
a nitrogenous base ( PURINE or a PYRIMIDINE )
N.B. Base Pairing : A always bonds with T (or U), G with C
Chains of nucleotides (polynucleotides) formed by DEHYDRATION SYNTHESIS reaction between the phosphate group of one nucleotide and the hydroxyl group on the sugar of another
This bonding gives polynucleotides a defined polarity reflecting the component nucleotides
[Diagram of Phosphodiester bond]
Polynucleotides & Nucleic Acid Function
Polynucleotide chains provide the structural and functional basis for the encoding and decoding of genetic information.
The sugar phosphate backbone carries a sequence of bases that makes up the genetic code as a series of triplet codons
Complementary base pairing holds the key to copying genetic information in the processes of DNA replication and transcription
The bases fit together A-T(U) and G-C are joined together by HYDROGEN BONDING
[Base pairing diagram]
A double stranded helix composed of two polynucleotide chains that run in opposite directions (anti-parallel)
The bases fit across the right-handed helix; one purine pairing with its complementary pyrimidine
The helix is the only shape that accommodates the purine-pyrimidine base pair and maintains stable hygrogen bonds
3 types of RNA which are SINGLE stranded but can fold to give 3D shapes or conformations:
mRNA - contains information transcribed from a DNA molecule and transports it to a ribosome
tRNA - collects amino acids and transports them to a ribosome to be fitted according to the messenger RNA code
rRNA (ribosomal RNA) - provides a major structural support component of the ribosome
A polymerase is an enzyme whose central function is associated with polymers of nucleic acids such as RNA and DNA
These are necessary for the following processes:
1) DNA REPLICATION: enables a complete copy of the genome to be passed on to each daughter cell during mitosis 2) TRANSCRIPTION OF DNA into RNA: : mechanism by which genes are expressed DNA polymerase
This enzyme forms phosphodiester bonds which are used to join DNA molecules or fragments together to produce recombinant DNA (recDNA)
Both polymerases,ligases and restriction endonucleases (cut DNA) are important components of a genetic engineer’s ‘toolkit’. They are used to manipulate DNA
The cell membrane/plasma membrane represents the barrier that separates the cell’s contents from the surrounding environment and controls what moves in and out
In eukaryotic cells, membranes are also used to generate compartments within the cell, each with a specialised function e.g. golgi apparatus, endoplasmic reticulum, lysosomes etc
Provides selectively permeable barriers
Localises reactions in the cell
Transport of solutes often against the concentration gradient (active transport)
Signal transduction – receptor proteins on the membrane surface recognise and respond to different stimulating molecules, enabling specific responses to be generated within the cell
Cell to cell recognition – the external surface of the membrane is important as it represents the cell’s biochemical “personality”. In multicellular organisms this allows cells to recognise each other as similar or different, which is necessary for the correct association of cells during development.
The basic composition and structure of the plasma membrane is the same as that of the membranes that surround organelles and other subcellular compartments.
The foundation is a phospholipid bilayer – polar hydrophilic heads on the outer surface and hydrophobic non-polar fatty acid tails form the inner surface. The membrane as a whole is often described as a fluid mosaic – a two-dimensional fluid of freely diffusing lipids, dotted or embedded with proteins which may function as channels or transporters across the membrane, or as receptors.
The Plasma Membrane
The idea that membranes were composed of phospholipids was first put forward in 1925. The currently accepted model for membrane structure was proposed by S.J. Singer (1971) as a lipid protein model and extended to include the fluid character in a publication with G.L. Nicolson in "Science" (1972)
The fluid mosaic model has 2 components, lipids and proteins. The lipids form the matrix bilayer of the membrane and the proteins carry out all of its functions
The membrane is not a static rigid structure, but a dynamic arrangement of lipids and proteins that drift laterally within it.
Types of Membrane Proteins
Proteins make up approximately 50% of the mass of the plasma membrane and can be classified into different groups depending on their arrangement in the membrane and/or their function
Proteins may be embedded in the lipid bilayer or attached to the surface
The embedded or INTRINSIC proteins may be transmembrane proteins (span the bilayer) or they may be linked to lipids on one side of the bilayer only
The peripheral or EXTRINSIC proteins are loosely attached to the membrane by ionic association with other proteins
Glycoproteins have carbohydrates attached to their extracellular domains.
Functions of Membrane Proteins
The main functions of these membrane proteins are as follows:
1) TRANSPORT PROTEINS
Transport non-diffusable substances across the membrane. May be either:
(a) Channel proteins – provide a ‘pore’ across the membrane through which molecules (usually small and charged) can diffuse
(b) Carrier proteins – these are more specific with binding sites for only one solute
Both these proteins permit passive transport (with a concentration gradient this is called facilitated diffusion)
To transport molecules against the concentration gradient, special types of the carrier proteins are needed. These harness energy to drive the transport process during active transport e.g. sodium-potassium pump
2) CELL RECOGNITION PROTEINS
The carbohydrate chain of the glycoprotein projects out of the cell enabling cell to cell recognition and serving as a cell “fingerprint”
Therefore, the immune system can recognise it’s own cells and organs e.g. ABO blood group antigens:
A = glycoprotein antigen A
O = no glycoprotein antigens
3) RECEPTOR PROTEINS
These have a specific conformation (shape) that allows binding of a particular molecule (the ligand)
The binding of the ligand will then trigger a response in the cell
A protein that catalyses a specific reaction
Some receptor proteins have enzymatic activity; the cytoplasmic portion of the protein catalyses a reaction in response to binding a ligand
5) INTRACELLULAR JUNCTIONS
Interactions between the plasma membranes of different cells is a frequent occurrence and takes place at cell junctions e.g.
-> In PLANTS
PLASMODESMATA – although each plant cell is encased in a boxlike cell wall, fine strands of cytoplasm, called plasmodesmata , extend through pores in the cell wall connecting the cytoplasm of each cell with that of its neighbors allowing direct exchange of materials
In ANIMALS , there are 3 types…
Spot desmosome – dense protein deposits that hold adjacent cells together by rivets. Mechanical strength is provided by the intracellular filaments passing from one desmosome to another
Tight junction – adjacent membrane proteins are bonded together preventing movement of materials in the space between the cells e.g. between epithelial cells lining the small intestine
Gap junction – doughnut shaped proteins from each cell joined together to form tiny channels allowing the passage of small molecules such as ions, amino acids and sugars
The eukaryotic cell is a 3D structure. It has a cytoskeleton anchored to proteins in the plasma membrane
These proteins both maintain shape and allow movement
The cytoskeleton is a dynamic structure, as the microfilaments and microtubules can depolymerise and repolymerise very easily
The cytoskeleton is made up of 3 components, in order of increasing diameter. They are …
1) Actin filaments/microfilaments
2) Intermediate filaments
These are composed of actin (protein)
They are arranged as 2 strands of protein molecules twisted together to give a rope-like structure about 7nm in diameter
These are present throughout the cell but are most highly concentrated just inside the plasma membrane
They are important in all cell movement and contraction
Actin fibres in a cell stained with a fluorescent strain specific for actin
2) Intermediate Filaments
These are about 10nm in diameter and are composed of tough fibrous protein strands twisted together
They are very stable structures in the cell and provide mechanical strength to animal cells which lack the strong cell walls of plants
Intermediate filaments can be anchored between the membrane to provide extra support
The nucleus in epithelial cells is held within the cell by a basketlike network of intermediate filaments made of keratins which have been stained here using a fluorescent stain
These are hollow tubes (like straws) composed of tubulin protein (a globular protein)
The tubulin protein subunits of microtubules associate in a cylindrical arrangement to generate the final microtubule - a relatively rigid structure
Microtubules only form around a centrosome (organising centre)
The centrosome provides a “nucleus” from which the microtubules form. These are important in cell division as part of the spindle fibre network and can move components within the cell
Microtubules growing in vitro from an isolated centrosome
But the primary importance of the cytoskeleton is in cell motility . The cytoskeleton extends throughout the cytoplasm and determines the internal movement of cell organelles, as well as cell locomotion and muscle fibre contraction
All of these components give mechanical support and shape to the cell
MOLECULAR INTERACTIONS IN CELL EVENTS
Molecular Interactions In Cell Events
- The vast number of coordinated and complex biochemical reactions that occur in an organism is summarised as the cell METABOLISM
- The reactions are in ordered pathways, controlled at each stage by ENZYMES
- Through these metabolic pathways, the cells are able to transform energy, breakdown macromolecules and synthesise new organic molecules needed for life
Uses energy to SYNTHESISE large molecules from smaller ones e.g.
Also known as endothermic reactions
These release energy through the BREAKDOWN of large molecules into smaller units e.g.
ATP -> ADP + Pi
Also known as exothermic reactions
Enzymes are commonly named by adding a suffix "-ase" to the root name of the substrate molecule it is acting upon. For example, Lipase catalyzes the hydrolysis of a lipid triglyceride. Sucrase catalyzes the hydrolysis of sucrose into glucose and fructose
A few enzymes discovered before this naming system was devised are known by common names e.g. pepsin, trypsin, and chymotrypsin which catalyse the hydrolysis of proteins
Enzymes are also given a standard reference number (European Commission Number) to help characterise the 1500 or so enzymes
ENZYMES These catalyse a transfer of a phosphate group onto a molecule such as a carbohydrate or protein Kinases To hydrolyse ATP. Many proteins have an ATPase activity which is essential for their function ATPases To hydrolyse phosphodiester bonds Nucleases To hydrolyse peptide bonds to breakdown proteins -> amino acids Proteases FUNCTION NAME
Form & Function of Enzymes
Enzymes work by bringing about substrate(s) of a reaction close together in an active site so that bond breakage or formation occurs at atomic level
This is often facilitated (helped) by specific chemical effects such as the transfer of proteins or the alteration of charge distribution around the target atoms
The substrate and enzyme must fit together very precisely
The Catalytic Cycle
A cycle of events that describes an enzyme combining with a substrate, remaining unchanged by the reaction and being available at the end of the reaction to combine with another substrate molecule
The Catalytic Cycle of Sucrase
Sucrase catalyses the hydrolysis of sucrose into it’s component monosaccharides, GLUCOSE & FRUCTOSE
1) At the start of the cycle, enzyme ( E ) and substrate ( S ) are available
2) The molecular interaction of enzyme and substrate at the active site forms the enzyme:substrate complex ( ES )
3) Catalysis occurs, forming the enzyme:product complex ( EP )
4) Products are released, leaving the enzyme free for the next substrate molecule
E S ES EP
Model for Enzyme Action
A common model for enzyme action is the lock and key hypothesis
However, this model is a little misleading in that it tends to give the impression that enzymes are rigid structures, whereas in fact, they are quite flexible and can alter their conformation in response to the binding of other molecules
The currently accepted model for enzyme action is the INDUCED FIT MODEL , in which conformational changes to the protein occur on binding of a substrate
The Induced Fit Model
The enzyme, HEXOKINASE , catalyses the transfer of a phosphate from ATP onto glucose
The active site and the two domains of the single polypeptide chain are clearly visible in the view of the backbone of the molecule
Think of the protein about to close around the substrate in the active site similar to the way your hand would close around a door handle
The effect of this is that glucose fits the active site more closely, and the binding of ATP is also enhanced
[see diagram of ‘The catalytic cycle of hexokinase]
Control of Enzyme Activity
The activity of enzymes must be reguated in some way to avoid metabolic chaos
Regulation can be achieved through a number of different mechanisms
A major influence is the NUMBER OF ENZYMES MOLECULES in the cell, which is controlled at the level of gene expression
COMPARTMENTALISATION also enables the cell to keep sets of enzymes together and away from other enzymes
TEMPERATURE & pH also affect enzyme activity
Many enzymes also require CO-FACTORS to function
However, the most effective way of enabling a fine control of enzyme activity is to alter the shape of the enzyme itself, and thus cause a change in its catalytic efficiency
Examples of this type of metabolic control include INHIBITORS, ALLOSTERIC EFFECTORS , COVALENT MODIFICATION and END-PRODUCT INHIBITION
Enzymes reaction rates can be changed by competitive inhibition and non-competitive inhibition
Inhibitors can be either competitive or non-competitive
COMPETITIVE inhibitors compete for the active site of the enzyme, thus reducing its effectiveness
competitive inhibitors are usually similar in structure to the substrate and the enzyme is ‘fooled’ into accepting the inhibitor, which blocks the active site
E.G: An example for competitive inhibition is the enzyme succinate dehydrogenase by malonate . Succinate dehydrogenase catalyses the oxidation of succinate to fumarate .
NON-COMPETITIVE inhibitors bind at a different location and change the conformation of the enzyme, thus altering the shape of the active site and again reducing the catalytic efficiency
Inhibition can either be reversible or non-reversible depending on how the inhibitor binds to the enzyme
Some inhibitors bind irreversibly with the enzyme molecules, inhibiting the catalytic activities permanently. The enzymatic reactions will stop sooner or later and are not affected by an increase in substrate concentration. These are irreversible inhibitors .
Examples are heavy metal ions including silver , mercury and lead ions.
These are enzymes that ‘change shape’ in response to the binding of a regulating molecule (often called a modulator or effector )
Allosteric modulators can be either positive or negative effectors of enzyme activity
They function by binding to allosteric sites that are distinct from the active site of the enzyme
Non-competitive inhibition is a form of allosteric regulation
In multi-subunit enzymes, the structure is more complex, and the enzyme often exists in 2 different conformational states:
ACTIVE and INACTIVE
These can be stabilised by binding the modulator
Positive modulators stabilise the active form of the enzyme
Negative modulators stabilise the inactive form
In addition to these modulators changing the activity of allosteric enzymes, sometimes the binding of the substrate itself to one active site enhances binding at the other active sites. This is known as COOPERATIVITY
Covalent modification of enzymes is another strategy used widely in metabolic regulation
One of the most common modifications is the addition of a PHOSPHATE group, which can alter the shape of a protein by attracting positively charged R-groups [phosphates carry 2 negative charges on the 2 single-bonded O atoms]
PROTEIN KINASES add phosphate groups and PHOSPHATASES remove them, thus the effect can be REVERSED
Some proteins are activated by phosphorylation , others are inactivated
An example of phosphorylation activating an enzyme is the skeletal muscle enzyme GLYCOGEN PHOSPHORYLASE
This enzyme releases glucose molecules from glycogen when heavy demands are placed on muscle tissue
This process is highly regulated. Traffic of sugar into and out of storage in glycogen is used to control the level of glucose in the blood, so glycogen phosphorylase must be activated when sugar is needed and quickly deactivated when glucose is plentiful
Glycogen phosphorylase is present as an inactive non-phosphorylated form which is converted to the active phosphorylated form by the addition of a phosphate group to a serine residue in the protein by the enzyme PHOSPHORYLASE KINASE
When the demand for glucose drops, PHOSPHORYLASE PHOSPHATASE removes the phosphate group and inactivates the enzyme
However … glycogen phosphorylase is also regulated by an allosteric effect !
Glucose and ATP act as negative modulators and AMP (adenine monophosphate) acts as a positive modulator – also causing the enzyme to shift to the active conformation
This is useful, because AMP is a product of ATP breakdown and will be more plentiful when energy levels are low and more glucose is needed
A further complication is that there is a hormonal control mechanism by adrenaline and glucagon
Another form of control by a covalent activating mechanism is proteolytic cleavage as found in the enzyme TRYPSIN
Trypsin is synthesised in the pancreas, but not in its active form as it would digest the pancreatic tissue
Therefore it is synthesised as a slightly longer protein called TRYPSINOGEN , which is inactive
Activation occurs when trypsinogen is cleaved by a protease in the duodenum
Once active, trypsin can activate more trypsinogen molecules, resulting in an autocatalytic cascade that produces a large number of active trypsin molecules very rapidly
Metabolism is organised as a series of metabolic pathways, and control of these pathways is an important feature of cell biochemistry
One way in which control can be exercised is END-PRODUCT INHIBITION
End-product inhibition is energetically efficient as it avoids the excessive (and wasteful) production of the intermediates of a pathway
This is a form of NEGATIVE FEEDBACK
For example, in the production of the amino acid isoleucine in bacteria, the initial substrate is threonine which is converted by five intermediate steps to isoleucine. As isoleucine begins to accumulate, it binds to an allosteric site of the first enzyme in the pathway thereby slowing down its own production. In this way, the cell does not produce any more isoleucine than is necessary.
The Sodium-Potassium Pump (Na+/K+ - ATPase)
A specific case of active transport
This is one of the best examples of active transport in animal cells
This pump transports Na+ ions out of the cell and K+ ions into the cell. Thus keeping the intracellular concentration of Na low compared to outside, and the intracellular concentration of K high
The pump is driven by hydrolysis of ATP
It uses about 30% of the energy available to any one animal cell!
The pump is a transmembrane carrier protein made up of 4 subunits (2 large and 2 small)
Structure: has 3 binding sites for sodium ions, 2 binding sites for potassium ions and a phosphorylation site to accept a phosphate from ATP
2 different conformations of protein are possible. This is controlled by the phosphorylation state of the protein
Hydrolysis of one ATP molecule fuels the export of 3 Na+ ions and the import of 2 K+ ions
Can work as fast as 300 Na+ ions per second if required!
[Sodium-Potassium Pump Diagram]
Sodium Potassium Pump Animation
Cell Signaling Molecules
Although cells can act as self-contained units, they don’t exist in isolation
Even a unicellular organism must detect and respond to outside influences e.g. chemicals , light and other cells
In a multicellular organism, the organisation of tissues and systems brings more complexity
Therefore, it is essential that cells can COMMUNICATE to enable their activities to be fully coordinated
Communication involves transmitting and receiving information
A SIGNALING cell sends a signal and is received by a TARGET cell
[ S ignal molecules can induce different responses in their target cells e.g. acetylcholine : causes cardiac muscle to relax, but skeletal muscle to contract ]
If a change in the form of a signal is required, it is called a SIGNAL TRANSDUCTION
Analogy: Faxing a letter – conversion of a printed form of information into an electronic form – back into a printed form ANIMATION
Communication Systems Signal molecules in the plasma membrane of the signal cell interact with membrane bound receptors on the target cell. These signals are therefore restricted to cells which are in direct contact CONTACT DEPENDENT Nerve cells or neurones elicit responses by the release of a neurotransmitter at synapses. Can signal over very long distances via a network of nerve cells. Very fast signalling e.g. GABA (Gamma-Amino-Butyric-Acid – an inhibitory neurotransmitter) NEURONAL Secretion of a local mediator. This affects cells in the immediate area of the signalling cell e.g. Histamine PARACRINE Secretion of a hormone into the bloodstream for dispersal. The signalling cell and the target cell can be far apart. Very slow method e.g. Insulin, Adrenaline ENDOCRINE
Extracellular HYDROPHOBIC Signaling Molecules
Some small hydrophobic molecules can cross the plasma membrane and enter the cell by diffusion
Best known classes are the STEROID hormones e.g. cortisol & testosterone and the THYROID hormones e.g. thyroxine
The hormones can diffuse across the plasma membrane and bind to receptor proteins that are located either in the cytosol or in the nucleus itself
They work by activating GENE REGULATORY PROTEINS in the cell, which stimulate transcription of particular sets of genes in the nucleus
The mode of action of cortisol: Cortisol is a steroid hormone that is released in the body in response to physical or psychological stress. The secretion of cortisol induces energy-directing processes for the purpose of providing the brain with sufficient energy sources that prepare an individual to deal with stressors. In addition to its role as a so-called "stress hormone", cortisol plays many key roles in almost every physiologic system. Regulation of blood pressure, cardiovascular function, carbohydrate metabolism, and immune function are among the best known functions of cortisol.
Action of Cortisol animation [Diagram]
Extracellular HYDROPHILIC Signaling Molecules
In contrast to the hydrophobic signals, the majority of signaling molecules are either too LARGE or too HYDROPHILIC to cross the plasma membrane
The receptor proteins for these signals must therefore present a binding site to the extracellular environment and elicit a response in the cytosol
There are 3 main classes of these cell surface transmembrane receptors all of which bind extracellular signal molecules, but generate intracellular responses in DIFFERENT ways …
1) ION-CHANNEL LINKED Receptor
These are also known as chemically-gated ion channels
They open pores through the protein in response to binding of a signal molecule
Ions flow through this ’gate’ generating an electrical effect
This type of receptor is found in excitable cells such as nerve and muscle cells
A neurotransmitter ( e.g. acetylcholine , noradrenaline ) binds to this type of receptor, altering its conformation to open or close a channel (often through or near the receptor) to the flow of Na2+, K+, Ca2+, or Cl- ions across the membrane.
Driven by their electrochemical gradient ( i.e. one side of the membrane has numerous ions, while the other side has few) the ions rush into or out of the cell, creating a change in the membrane potential due to the positive or negative nature of the ions.
This flow of ions through the channel can trigger a nerve impulse, or alternatively stop one from occurring.
2) ENZYME LINKED Receptor
Found in all types of cells
Generate an enzyme activity (usually a KINASE activity) on the cytoplasmic end of the protein
This kinase activity causes the phosphorylation of other intracellular proteins, thereby activating them
3) G-PROTEIN LINKED Receptor
Activate a GTP-binding protein (the G-protein ) that sets off a chain of events in the cell
This group of receptors is the largest known, and many different signals and responses can be associated with G-protein activity
All have the same structural arrangement within the membrane – known as a seven-pass transmembrane protein
Several hundred types of receptor are known, which bind signals as diverse as peptide hormone, amino acids, fatty acids and neurotransmitters
On binding the signal, the G-protein is activated by the binding of GTP
This activated protein diffuses away from the receptor protein site and activates its target protein
This may be an ion-channel protein or an enzyme such as adenylate cyclase or phospholipase C These enzymes catalyse the formation of small molecules known as secondary messengers which trigger the intracellular response to the original signal transduction event to the cell surface.
Second messengers are important parts of the signal transduction pathway, and can have many different effects
An outline of the cAMP pathway is shown below:
[Insert cAMP pathway diagram]
Very complex area !
Signals can be of many different types and can act either by diffusing across the plasma membrane (such as STEROID HORMONES e.g. testosterone and NITRIC OXIDE ) or by interacting with a receptor protein on the cell surface
The variety of signals, receptors and responses means that the system of signal reception and transduction can generate very specific effects in different types of cell
The response of a cell to a signal can involve ion flow, activation of specific proteins, or changes in gene expression
These effects can be short-lived, as in the case of the generation of an action potential, or they may be permanent alterations that control the developmental fate of the cell
It is therefore clear that the idea of a cell as a self-contained unit is in fact very far from the reality of the situation - cells are constantly engaged in the exchange of information in the form of molecular signals and it is this that enables cells in multicellular systems to function in an integrated way.
Quiz 1 Quiz 2
APPLICATIONS OF DNA TECHNOLOGY One of the defining features of modern biology is the extensive use of the technology of gene manipulation . It is now possible to manipulate DNA directly to produce recombinant DNA . The manipulated genes can be replaced back into the original, or a different, organism to produce transgenic plants and animals.
Applications of DNA Technology
1) The Human Genome Project: genetic mapping, DNA sequencing, genome analysis/comparison
2) Human Therapeutics: detecting genetic disorders, gene therapy
3) Forensic Uses: DNA profiling
4) Agriculture: transgenic plants, production of BST
The Human Genome Project
The genome of an organism is it’s complete complement of genetic information
Completed in 2003, the international Human Genome Project was a 13-year project coordinated by the U.S. Department of Energy and the National Institutes of Health.
Project goals were to …
- identify all the approximately 20,000-25,000 genes in human DNA,
- determine the sequences of the 3 billion chemical base pairs that make up human DNA
- store this information in databases
- improve tools for data analysis
- address the ethical, legal, and social issues that may arise from the project.
The human genome project has been achieved using 3 approaches:
a) GENETIC MAPPING
b) PHYSICAL MAPPING
c) DNA SEQUENCING
Firstly however, the desired DNA sequences must be amplified. The process used to do this is the POLYMERASE CHAIN REACTION (PCR)
The Polymerase Chain Reaction (PCR)
Polymerase chain reaction ( PCR ) is a revolutionary molecular biology technique for enzymatically replicating DNA
The technique allows a small amount of the DNA molecule to be amplified many times in an exponential manner
PCR is commonly used in medical and biological research labs for a variety of tasks, such as the detection of hereditary diseases, the identification of genetic fingerprints, the diagnosis of infectious diseases, the cloning of genes, and paternity testing.
PCR product compared with DNA ladder in agarose gel
DNA ladder ( lane 1 ),
the PCR product in low concentration ( lane 2 ),
and high concentration ( lane 3 ).
Stages in PCR
PCR, as currently practiced, requires several basic components. These components are:
DNA template, which contains the region of the DNA fragment to be amplified
Two primers , which determine the beginning and end of the region to be amplified ( primers = short lengths of a known DNA sequence )
DNA-Polymerase , which copies the region to be amplified
Nucleotides , from which the DNA-Polymerase builds the new DNA
Buffer, which provides a suitable chemical environment for the DNA-Polymerase
The PCR reaction is carried out in a thermal cycler . This is a machine that heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction.
The PCR process consists of a series of twenty to thirty cycles. Each cycle consists of three steps:
(1) The double-stranded DNA has to be heated to 94-96°C in order to separate the strands. This step is called denaturing; it breaks apart the hydrogen bonds that connect the two DNA strands.
(2) After separating the DNA strands, the temperature is lowered so the primers can attach themselves to the single DNA strands. This step is called annealing. The temperature of this stage depends on the primers and is usually 5°C below their melting temperature (45-60°C)
(3) Finally, the DNA-Polymerase has to fill in the missing strands. It starts at the annealed primer and works its way along the DNA strand. This step is called extension. The extension temperature depends on the DNA-Polymerase
Taq DNA Polymerase: This is a thermal stable enzyme isolated from thermophilic bacteria. This enzyme canonly synthesis DNA in one direction 3’ - 5’
Applications of PCR
1 ) MOLECULAR BIOLOGICAL RESEARCH - gene screening analysis (looking for a gene) and DNA cloning (copying particular DNA sequences)
2) GENETIC MAPPING STUDIES e.g. human genome project, sequence tagging on genome sites
3) CLINICAL & DIAGNOSTIC USES - screening and diagnosis of HIV; cancer (detects mutations of oncogenes); genetic disorders e.g. cystic fibrosis
4) GENETIC IDENTIFICATION and DNA TYPING - forensic and parentage testing; sex determination of pre-natal cells; classification of species
5) IDENTIFICATION of TRACE AMOUNTS of DNA - detection of contamination of foodstuff by: food-borne pathogens, genetically modified organisms in food products, presence of pork in beef etc
Nucleic Acid Hybridisation
Once a gene has been isolated from a complete genome as a piece of DNA; we may want to know from which chromosome gene it came from and where that chromosome is located; or from which cells of the organism the gene is transcribed; or to test a sample of human DNA for mutations in the gene suspected of causing an inherited disease
All these questions can be answered by taking advantage of the fundamental property of DNA :
COMPLEMENTARY BASE PAIRING
Remember, the 2 strands of DNA are held together by HYDROGEN BONDING . These bonds can be broken by heating to 90 o c or altering the pH
These treatments release the single strands but DO NOT break the strong covalent bonds that link the nucleotides together
If the process is reversed i.e. slowly lowering the temperature and bringing the pH back to normal, the complementary strands will reform double helices - this is known as HYBRIDISATION
Using this technique, particular DNA sequences can be identified by hybridisation with the aid of a NUCLEIC ACID PROBE
Nucleic acid hybridization
(A) If the DNA helix is separated into two strands, the strands should reanneal, given the appropriate ionic conditions and time.
(B) Similarly, if DNA is separated into its two strands, RNA should be able to bind to the genes that encode it. If present in sufficiently large amounts compared with the DNA, the RNA will replace one of the DNA strands in this region
A Nucleic Acid Probe - a short, single-stranded DNA or RNA molecule that has been radioactively labelled ( e.g. 32-phosphate 32 P ) and is used to identify a complimentary nucleic acid sequence
Genetic Linkage Mapping
Genetic maps are based on the recombination frequency between genetic markers during MEIOSIS [see Higher notes!]
These can be used to locate genes on particular chromosomes and establish the order of the genes and the approximate distance between them
This approach relies on having genetic markers that are detectable
Genetic markers are any gene that shows variation (different alleles). These include genes and other DNA sequences such as microsatellites , which are tandem repeats of units 2-4 bp in length. These units are also known as short tandem repeats and are distributed fairly evenly over the genome, and may even occur within genes.
Sometimes these are genes that cause disease, traced in a family by pedigree analysis
The marker alleles must be HETEROZYGOUS so that meiotic recombination can be detected
NB: if 2 genes are on different chromosomes - they are unlinked and will sort independently during meiosis
If 2 genes are on the same chromosomes they are physically linked and a crossover between them during Prophase I of meiosis can generate non-parental genotypes
The chance of a crossover occuring increases as linked genes become further apart. In fact, they may behave as if they are essentially unlinked
Genetic mapping is used to produce a picture of the locations of the marker loci on the chromosome. However, it doesn’t provide the precise distances between the genes
[Insert Genetic Linkage diagram]
A physical map is a more detailed map of a genetic map
As with genetic maps, construction of a physical map requires markers that can be mapped to an exact location on the DNA
Physical maps of the genome can be constructed in a number of ways, all of which aim to generate a map in which the distances between markers are known with reasonable accuracy
Fragments of DNA are made by cutting with restriction enzymes or endonucleases
These are enzymes that cleave DNA at certain nucleotide sequences, thereby generating specific fragments
The recognition sequences where restriction enzymes are short (4,5 or 6 base pairs long) sequences that occur at defined positions in the DNA
Using a combination of these enzymes and measuring the size of fragments produced, the ‘puzzle’ can be pieced together to give the pattern of restriction enzyme recognition sites in the DNA
Defined fragments can then be identified either by their size or using a specific DNA probe to bind to its complementary map [ electrophoresis or nucleic acid hybridisation ] diagram
Restriction Mapping : An example
The most straightforward method for restriction mapping is to digest samples of the DNA with a set of individual enzymes, and with pairs of those enzymes
The digests are then "run out" on an agarose gel to determine sizes of the fragments generated. If you know the fragment sizes, it is usually a fairly easy task to deduce where each enzyme cuts, which is what mapping is all about
Restriction Mapping : An example
To illustrate these ideas, consider a plasmid that contains a 3000 base pair (bp) fragment of unknown DNA. Within the vector, immediately flanking the unknown DNA are unique recognition sites for the enzymes Kpn I and BamH I. As illustrated in the diagram below, consider first separate digestions with Kpn I and BamH I :
Digestion with Kpn I yields two fragments: 1000 bp and "big". Since there is a single Kpn I site in the vector, the presence of a 1000 bp fragment tells you that there is also a single Kpn I site in the unknown DNA and that it is 1000 bp from the Kpn I in the vector. The "big" fragment consist of the vector plus the remaining 2000 bp of the unknown
Digestion with BamH I yields 3 fragments: 600, 2200 and "big". The "big" fragment is again the vector plus a little bit (200 bp in this case) of unknown DNA. The presence of 600 and 2200 bp fragments indicate that there are two BamH I sites in the unknown. You can deduce immediately that one BamH I site is 2800 bp (600 + 2200) from the BamH I in the vector. The second BamH I site can be in one of two positions: 600 or 2200 bp from the BamH I site in the vector
At this point, there is no way to know which of these alternative positions is correct
The trick to determining where the second BamH I site is located is to digest the plasmid with Kpn I and BamH I together
This so-called double digest yields fragments of 600, 1000 and 1200 bp (plus the "big" fragment). The 600 bp fragment is the same as obtained by digestion with BamH I alone. The 1000 and 1200 bp fragments tell you that Kpn I cut within the 2200 bp BamH I fragment observed when the plasmid was cut with BamH I alone
You already know where Kpn I cuts in the unknown DNA, and you therefore now know the location of the second BamH I site!
Used to locate genes or other DNA sequences on a physical map or to locate genes associated with disorders
The marker DNA and target DNA must be linked
DNA probes used to locate and isolate multiple copies of DNA that have complementary sequences of DNA to the probe in libraries
2 libraries are made, one from cloned fragments of the marker and one from cloned fragments of the target DNA
Different restriction enzymes are used so that the fragments in each library are different but overlap
Since nucleic acids are negatively charged, they migrate toward the positive pole in an electric field
When the electric field is applied through the gel, molecular sieving takes place. Shorter chains move faster than longer ones. Thus, the chains are spread out in the gel according to their size.
Double-stranded DNA can be visualized by adding ethidium bromide, a flat aromatic chemical that fits between base pairs in the double helix. Only when bound to DNA does the ethidium bromide fluoresce orange when irradiated with UV
The final stage of the genome project is to determine and assemble the actual DNA sequence itself. For this to happen:
DNA fragments must be generated
The sequencing technology must be accurate and fast
Computer hardware/software must be available to analyse the data
DNA Sequencing cont .
The technique used for sequencing is the Dideoxy Chain Termination method as developed by F. Sanger in the 1970’s
This method relies on making a copy of the chosen DNA template
[See Student monograph for a more comprehensive explanation – pg.154 - 157]
Comparative Genome Analysis
In addition to mapping the human genome, the genomes of other species are also being mapped. These include species important to biological research and agriculture such as the mouse, chicken, pig, cow, rice, wheat, Caenorhabditis elegans (nematode), Drosophila melanogaster (fruit fly), Saccharomyces cerevisiae (yeast), Escherichia coli , and other prokaryotes.
The genomes of some of these organisms, such as E. coli , yeast, the nematode and the fruit fly have now been completely mapped and sequenced. These maps can be used to locate homologous genes in the human genome and to help in determining gene function.
Comparative genome analysis is being used to find out more about evolution . The number of differences in an amino acid sequence can be used to calculate the time since two species diverged from a common ancestor. If there are lots of differences between the maps, it can be deduced that the species diverged longer ago than if there are only a few differences. This type of information is used alongside other methods of measuring the rate of evolution.
Gene maps can be used to predict gene order . If gene X is found next to gene Y and Z in one species, the likelihood is that it will be found next to the same two genes in another closely related species. Comparative maps will be used to find candidate genes for phenotypes mapped in species as diverse as chicken and human.
How is our knowledge of DNA technology being used
today in human therapeutics?
Congenital abnormalities are genetically based diseases and there therefore inherited
caused by a single gene defect ( Cystic Fibrosis, Sickle Cell Anaemia, Haemophilia ) caused by defects in several genes ( Heart Disease, Diabetes, Obesity, some cancers )
Detecting genetic disorders
Characteristics of a monogenic disease usually begins with
the presentation of disease symptoms
Step 1 : Trace disease through family using PEDIGREE ANALYSIS to determine if the faulty gene is dominant , recessive or X-linked (crucial for genetic counselling )
Step 2 : Genetic maps are used to identify genetic markers, co- inherited along with the disease. Recombination frequencies give the distance of the marker to the diseased gene. The marker is then located on a more detailed physical map . The gene is then tracked down, characterised and sequenced , leading to accurate diagnostic procedures and potential new treatments
Cystic Fibrosis (CF)
Duchenne Muscular Dystrophy (DMD)
X linked inheritance of Duchenne Muscular Dystrophy Autosomal Recessive Inheritance of Cystic Fibrosis
Using either CF or DMD as a case study, write a report about the discovery and treatment of the disease making sure to :
explain the genetic mutation involved
describe the methods/tests used to detect genetic disorders such as Cystic Fibrosis and Duchenne Muscular Dystrophy;
explain the importance of genetic counselling;
explain how gene therapy could be used to treat genetic disorders;
include some analysis of results of gene therapy trials;
discuss the legal, moral and ethical issues for the future
http://www.gig.org. uk & http://www.who. int /genomics/ elsi /en/
When a gene defect such as CF or DMD has been identified and the gene cloned, there is a possibility of using the ‘ good’ copy of the gene to overcome the problem
This is known as GENE THERAPY : The replacement of a ‘faulty’ gene with a ‘normal’ gene and/or the insertion of an extra gene with the intention that the gene product will play a therapeutic role
Therefore, the cause of the disease is targeted rather than the symptoms
Gene therapy can be used to …
add functioning genes to cells that have lost the gene
inhibit the spread of a virus e.g. by preventing DNA replication
kill abnormal human cells by inserting killer genes directly into a patient’s tumour (they will only kill the cells expressing those genes)
add genes that will specifically inhibit oncogenes or their products
add genes that make them more susceptible to chemo or radiotherapy
Gene therapy can only be used however, when the following conditions are met:-
1) The normal gene is available in a cloned form
2) The affected cells are accessible (either in vivo or ex vivo )
3) There are suitable vehicles for delivery of the gene (viral vectors, lyposomes, artificial DNA)
4) The gene functions normally in its target cells ( i.e. it produces functioning gene product)
Both CF and DMD are potential candidates for gene therapy. But in order for the genes to be dlivered to the correct target cells, suitable VECTORS must be used. To decide the vector, several factors must be decided upon:
efficiency of delivery to the target cell
specificity of the delivery to the target cell
whether the vector will provoke an immune response
the size of the DNA the vector can carry
stability and longevity once delivered
expression of the gene in the target cell
Ethical Issues in Gene Therapy
Are we PLAYING GOD?
The DNA of every individual is different unless you
are an identical twin. DNA technology has therefore
become one of the most important tools for identifying
individuals in both criminal cases and paternity disputes
What is DNA Fingerprinting?
The use of the the small stretches of DNA that vary among individuals to determine the probability of a match between samples from different people.
DNA fingerprinting is routinely used to identify potential suspects using DNA left behind at a crime scene. Police have created DNA Fingerprints from samples as diverse as chewing gum, cigarette butts, toenail clippings and even watch straps! DNA fingerprinting has also been used to help us …
… cook basmati rice to perfection
… monitor illegal trade in protected species
… confirm the authenticity of wine
Stages in generating a DNA profile
1) DNA isolation
2) Restriction enzyme digestion
3) Gel electrophoresis
4) Blotting DNA onto afilter
5) Hybridisation with a nucleic
A sample from the subject is matched with a referenced sample (DNA sample from crime scene or a relative in a civil case)
Limited samples of DNA are amplified using PCR
DNA profiling relies on repetitive, hypervariable DNA
FORENSICS : An example
A terrible crime has taken place. A middle-aged man was murdered during a burglary. Forensic scientists collected a blood sample from the victim and a sample from the scene of the crime. Blood samples were also taken from four suspects identified by the police. Forensic scientists have prepared the autoradiograph on next slide from DNA extracted from the blood samples. The prosecution wishes to use it as evidence in a court case
Which part of a blood sample is required for this analysis?
Who should be charged with the crime?
The prosecution wishes to use the forensic evidence in court but is concerned that the autoradiograph is not conclusive proof. Why?
How could the accuracy of the analysis be improved?
explain how and why a plasmid isolated from Agrobacterium is used in the production of transgenic plants;
· explain how foreign DNA is isolated and inserted into plasmids;
· explain how protoplasts are made;
· explain the use of protoplasts and selective media in the production of transgenic plants;
· describe the production of transgenic tomato plants;
· explain how plants resistant to insect damage can be made using bacterial toxin genes;
· discuss moral and ethical issues related to the use of transgenics;
· describe the production and use of cloned bovine somatotrophin (BST).
explain how and why a plasmid isolated from Agrobacterium is used in the production of transgenic plants;
explain how foreign DNA is isolated and inserted into plasmids
explain how protoplasts are made
explain the use of protoplasts and selective media in the production of transgenic plants;
discuss moral and ethical issues related to the use of transgenics;
Transgenic Tomato Plants: Flavr Savr
In 1994, the first “genetically modified” food was approved by the FDA to go to market. The tomato, Flavr Savr, was modified by Calgene (a biotechnology company) using antisense technology resulting in altered ripening. Traditional tomatoes must be picked from the vine while still green in order to maintain their firmness during transport to the supermarket. The tomatoes are then sprayed with ethylene, their natural ripening agent, in order to turn the tomatoes red. Flavr Savr tomatoes are designed so they can ripen on the vine longer while maintaining firmer skin, thus producing a fuller flavoured tomato on the supermarket shelves.
Public concern surrounded Flavr Savr’s introduction to the market. Debate raged across North America. How would this change the tomato? Was this tomato dangerous to our health? Should we have concerns about allergies? Nutrition? Toxins? What were the dangers to the environment? What about gene transfer across different organisms? Had Calgene created a “Frankenfood”?
Bovine Somatotrophin (BST)
Bovine somatotropin (bST) is a natural hormone that stimulates milk production. Biotechnology companies began manufacturing a genetically engineered version of bST in the early 1990s.
On November 5, 1993, the FDA approved genetically engineered bST for commercial use in the United States. Treating dairy cows with this hormone increases milk production by as much as 20 percent, and no detectable difference has been found between milk from treated cows and milk from untreated cows. The hormone bST has no adverse effects on the health of treated cows, and milk and meat from bST-treated cows are both safe for human consumption.
Scientists throughout the world—researchers working in academia, in government, and in the dairy industry—conducted more than 2,000 scientific studies of bST. The studies show clearly the efficacy, the safety, and the benefits that can be realized by integrating bST into dairy production technology. To stem the tide of misinformation about bST, the FDA itself—in an unprecedented move—sponsored a 1990 article in Science magazine stating that bST was perfectly safe.
But despite the scientific data and the proved efficacy of bST, opposition arose. One day before U.S. sales of milk from treated cows began, consumer activists dressed up in cow suits and dumped milk to protest the use of bST. Jeremy Rifkin, the president of the Foundation for Economic Trends, raised particularly vigorous objections to the introduction of bST.