Course Outline This course involves a detailed study of 4 Units: (1) Cell and Molecular Biology (2) Environmental Biology (3) Physiology, Health & Exercise (4) Investigation There is practical work within units (1) - (3), while unit (d) is based on research and experimentation entirely.

This course involves a detailed study of 4 Units:

(1) Cell and Molecular Biology

(2) Environmental Biology

(3) Physiology, Health & Exercise

(4) Investigation

There is practical work within units (1) - (3), while unit (d) is based on research and experimentation entirely.

Overview 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.

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

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 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

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

Internal Assessment 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

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%)

Career Opportunities 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

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]

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: Bacteria Archaea Plant cells Animal cells Prokaryotes and Eukaryotes are 2 distinct cell types with STRUCTURAL differences PROKARYOTES EUKARYOTES

Living organisms can be classified into 3 major domains:

Bacteria

Archaea

Plant cells

Animal cells

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. 1 um

Simply stated, prokaryotes are molecules surrounded by a membrane and cell wall.

Prokaryotes 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).

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 Cell Wall Contains PEPTIDOGLYCAN (only found in bacteria). Large complex molecule consisting of polysaccharide polymers cross-linked by short chains of amino acids Capsules Sometimes the cell wall is further surrounded by a gelatinous polysaccharide sheath called an attach CAPSULE , GLYCOCALYX or SLIME LAYER Plasma Membrane 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

Cell Wall

Contains PEPTIDOGLYCAN (only found in bacteria). Large complex molecule consisting of polysaccharide polymers cross-linked by short chains of amino acids

Capsules

Sometimes the cell wall is further surrounded by a gelatinous polysaccharide sheath called an attach CAPSULE , GLYCOCALYX or SLIME LAYER

Plasma Membrane

Basic structure of the phospholipid bilayer is the same for all bacteria

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.

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

These are produced by some bacteria to survive unfavourable environmental conditions. Dormant forms are metabolically inactive and only germinate under suitable conditions

Classifying Prokarotes 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

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

Eukaryotes 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 ! ADVANTAGES: 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

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

ADVANTAGES:

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

Eukaryotes The basic eukaryotic cell contains the following: - membrane-bound nucleus - plasma membrane - glycocalyx (components external to the plasma membrane) - 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

The basic eukaryotic cell contains the following:

- membrane-bound nucleus

- plasma membrane

- glycocalyx (components external to the plasma

membrane)

- 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]

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

Plasma Membrane Plasma Membrane A lipid/protein/carbohydrate complex, providing a barrier and containing transport and signalling systems.

Plasma Membrane

A lipid/protein/carbohydrate complex, providing a barrier and containing transport and signalling systems.

Nucleus Nucleus 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

Nucleus

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

Mitochondria Mitochondria 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.

Mitochondria

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. Ribosomes Protein and RNA complex responsible for protein synthesis

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.

Ribosomes

Protein and RNA complex responsible for protein synthesis

Golgi Apparatus Golgi apparatus 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.

Golgi apparatus

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 Centrioles Centrioles are found only in animal cells. They function in cell division.

Centrioles

Centrioles are found only in animal cells. They function in cell division.

Lysosymes Lysosymes A membrane bound organelle that is responsible for degrading proteins and membranes in the cell, and also helps degrade materials ingested by the cell.

Lysosymes

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 Peroxisomes or Microbodies Produce and degrade hydrogen peroxide, a toxic compound that can be produced during metabolism

Peroxisomes or Microbodies

Produce and degrade hydrogen peroxide, a toxic compound that can be produced during metabolism

Chloroplasts Chloroplasts 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.

Chloroplasts

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.

Vacuoles Vacuoles Membrane surrounded "bags" that contain water and storage materials in plants.

Vacuoles

Membrane surrounded "bags" that contain water and storage materials in plants.

Cell wall Cell wall Plants have a rigid cell wall in addition to their cell membranes. They provide support for the plant.

Cell wall

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

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

Main differences are STRUCTURAL :

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

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 .

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 Cycle 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

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:

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 .

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)

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 .

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 .

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 .

Interphase 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.

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.

Prophase 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.

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.

Metaphase 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.

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.

Anaphase 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.

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.

Telophase 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.

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.

Cytokinesis 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.

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.

Remember …! P rophase M etaphase A naphase T elophase C yto k inesis P ositive M ental A ttitude T owards C alvin K lein

P rophase

M etaphase

A naphase

T elophase

C yto k inesis

P ositive

M ental

A ttitude

T owards

C alvin K lein

DNA Replication 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 .

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 .

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]

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)

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.

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

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

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

Causes of Cancer Somatic Cell mutations Proliferation genes (proto-oncogenes -> oncogenes) Anti-proliferation genes (also known as Tumour-suppressor genes)

Somatic Cell mutations

Proliferation genes (proto-oncogenes -> oncogenes)

Anti-proliferation genes (also known as Tumour-suppressor genes)

Mitotic Index Fraction or percentage of cells in a given sample that contain condensed chromosomes i.e. the cells are undergoing mitosis and dividing http://www-saps.plantsci.cam.ac.uk/worksheets/scotland/mitosis.htm

Fraction or percentage of cells in a given sample that contain condensed chromosomes i.e. the cells are undergoing mitosis and dividing

GLOSSARY 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.

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 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.

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.

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 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!

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!

Cellular Differentiation 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

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

Cellular Differentiation 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.

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.

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 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

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

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

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: Growth medium Type of growth container or fermenter Temperature pH Gas exchange Aseptic conditions Method for monitoring cell growth Safety measures and implications

In order for cells to grow, the conditions must be

just right for each cell type. The cytologist must

therefore consider the following:

Growth medium

Type of growth container or fermenter

Temperature

pH

Gas exchange

Aseptic conditions

Method for monitoring cell growth

Safety measures and implications

Aseptic conditions 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

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:

Microorganisms 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

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

There are 2 recognised categories of micro-organism:

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]

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

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 the by-products

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

the by-products

Microbial Growth Culture Requirements Important factors that must always be considered are: the nutrient media temperature pH gaseous environment light 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

Important factors that must always be considered are:

the nutrient media

temperature

pH

gaseous environment

light

Nutrient Media 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

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

Nutrient Media 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 !!!

There are 2 types of media commonly used:

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

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

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

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 Contains … - mixture of glucose , amino acids , salts , water and antibiotics - 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 *

Contains …

- mixture of glucose , amino acids , salts , water

and antibiotics

- 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

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 ...

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

(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

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]

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]

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

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

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

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 !

Totipotency All plant cells are totipotent - they each have the ability to express the full genetic potential of that 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

Introduction Living systems are composed of a limited number of elements namely… CARBON, HYDROGEN, OXYGEN, NITROGEN, PHOSPHORUS & SULPHUR 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

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

Polymers 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 Dehydration Hydrolysis

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

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

Carbohydrates 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

Composed of CARBON, HYDROGEN & OXYGEN

Glucose (C 6 H 12 O 6 ) Hexose sugar 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 )

Hexose sugar

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

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

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 ANIMATION

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

Polysaccharides 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

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

1. Starch 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

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

2. Glycogen 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 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

3. Cellulose 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

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

4. Chitin A homopolysaccharide similar to cellulose in structure. Component of many insect exoskeletons - very strong and rigid; also resistant to chemicals. 5. Glycosaminoglycans A heteropolymer found in skin and connective tissue of vertebrates

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

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 Thermal Insulation

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

Thermal Insulation

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

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: 1. Triglycerides Most common type of lipid 3 fatty acids and a glycerol molecule are linked by an ester bond formed during dehydration synthesis 2. Phospholipids 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 3. Steroids Very different structure – 4 carbon rings with variety of different side chains

3 types of lipids which are important to cells:

1. Triglycerides

Most common type of lipid

3 fatty acids and a glycerol molecule are linked by an ester bond formed during dehydration synthesis

2. Phospholipids

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

3. Steroids

Very different structure – 4 carbon rings with variety of different side chains

Triglycerides cont. 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 ANIMATION

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

Phospholipids 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

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

Functions of Phospholipids Essential components of cells and organelle membranes Components of lung surfactants

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

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 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

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. R GROUP 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

At neutral pH, amino acids exist in ionised forms. Once joined, the charges on amino acids disappear.

R GROUP

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

Types of Amino Acids - H Gly/G Glycine Non-polar - CH 2 OH Ser/S Serine Uncharged Polar - (CH 2 ) 4 NH 4 Lys/K Lysine Basic - CH 2 COOH Asp/D Aspartic Acid Acidic R-Group Abbreviations Name Amino Acid Class

The Peptide Bond 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

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 ANIMATION

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

Protein Structure 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: Non-covalent bonds Hydrogen Bonds Ionic bonds Van der Waals interactions Hydrophobic interactions between R groups

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:

Non-covalent bonds

Hydrogen Bonds

Ionic bonds

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

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

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)

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

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

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

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

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

Alpha Helix 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

Alpha Helix

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

Beta sheet 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

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

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]

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]

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

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 )

Phosphodiester Bond 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]

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]

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]

DNA 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

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

RNA 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

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

Polymerase enzymes 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

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:

DNA Ligase 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

This enzyme forms phosphodiester bonds which are used to join DNA molecules or fragments together to produce recombinant DNA (recDNA)

Cell Membranes 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

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

Membrane functions Provides selectively permeable barriers Compartmentalisation 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.

Provides selectively permeable barriers

Compartmentalisation

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.

Membrane Structure 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 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.

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.

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: Transport Cell recognition Receptor sites Enzymes Intracellular Junctions

The main functions of these membrane proteins are as follows:

Transport

Cell recognition

Receptor sites

Enzymes

Intracellular Junctions

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

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 Usually glycoproteins 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

Usually glycoproteins

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

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

4) ENZYMES 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

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

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

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 Cytoskeleton 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 MICROFILAMENTS MICROTUBULES INTERMEDIATE FILAMENTS

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 The cytoskeleton is made up of 3 components, in order of increasing diameter. They are … 1) Actin filaments/microfilaments 2) Intermediate filaments 3) Microtubules

The cytoskeleton is made up of 3 components, in order of increasing diameter. They are …

1) Actin filaments/microfilaments

2) Intermediate filaments

3) Microtubules

1) Microfilaments 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

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

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 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

3) Microtubules 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

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

Functions 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

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 CATALYSIS : - 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

CATALYSIS :

- 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

Anabolic Reactions Uses energy to SYNTHESISE large molecules from smaller ones e.g. Amino Acids->Proteins Also known as endothermic reactions ENDOTHERMIC REACTION

Uses energy to SYNTHESISE large molecules from smaller ones e.g.

Amino Acids->Proteins

Also known as endothermic reactions

Catabolic Reactions These release energy through the BREAKDOWN of large molecules into smaller units e.g. Cellular Respiration: ATP -> ADP + Pi Also known as exothermic reactions EXOTHERMIC REACTION

These release energy through the BREAKDOWN of large molecules into smaller units e.g.

Cellular Respiration:

ATP -> ADP + Pi

Also known as exothermic reactions

Naming Enzymes 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 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

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

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

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

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

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]

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

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

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

Inhibitors Enzymes reaction rates can be changed by competitive inhibition and non-competitive inhibition Inhibitors can be either competitive or non-competitive

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

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 .

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

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.

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.

Allosteric Enzymes 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

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

Allosteric Enzymes

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

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 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

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

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 !

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

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

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

Proteolytic Cleavage 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

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

End-Product Inhibition 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

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)

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

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 Cell-cell recognition 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

Cell-cell recognition

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 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

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

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]

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 …

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

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.

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

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

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. G-Protein animation

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.

G-Protein animation

The cyclic AMP (cAMP) signal transduction pathway Adenylate cyclase activity generates cyclic AMP (cAMP), phospholipase C generates Inositol Triphosphate (IP3) 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]

Adenylate cyclase activity generates cyclic AMP (cAMP), phospholipase C generates Inositol Triphosphate (IP3)

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]

Signal Transduction 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

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.

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

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 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 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. [Insert diagram]

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.

[Insert diagram]

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 ).

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.

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’

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

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

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

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 :

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

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

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

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

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]

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]

Physical Mapping 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

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

Restriction Mapping 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

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

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 :

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

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!

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

Chromosome Walking 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

Chromosome Walking

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

Gel Electrophoresis 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

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

DNA Sequencing 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

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]

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.

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.

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.

Human Therapeutics How is our knowledge of DNA technology being used today in human therapeutics? Congenital abnormalities are genetically based diseases and there therefore inherited 2 types: MONOGENIC : POLYGENIC : caused by a single gene defect ( Cystic Fibrosis, Sickle Cell Anaemia, Haemophilia ) caused by defects in several genes ( Heart Disease, Diabetes, Obesity, some cancers )

How is our knowledge of DNA technology being used

today in human therapeutics?

Congenital abnormalities are genetically based diseases and there therefore inherited

2 types:

MONOGENIC :

POLYGENIC :

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

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

Mini-project 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/

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/

Gene Therapy 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

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

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)

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

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? http://www.gig.org. uk

Are we PLAYING GOD?

http://www.gig.org. uk

Forensic Uses 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

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

DNA Fingerprinting 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

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 acid probe 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

1) DNA isolation

2) Restriction enzyme digestion

3) Gel electrophoresis

4) Blotting DNA onto afilter

5) Hybridisation with a nucleic

acid probe

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

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

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

Whodunnit ? 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?

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?

Agriculture 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;

· 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).

Transgenic Plants  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;

 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”?

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.

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.

Issues surrounding transgenics