the cell

Introduction to Molecular Biology

What is Molecular Biology?

Study of biology at molecular level

Basic Molecular Biology

Applied Molecular Biology

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Which Molecular Level?

• Nucleic Acids

DNA

RNA

• Proteins

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What to know about nucleic acids?

• Structure

Chemical properties

Physical properties

Types

• Synthesis

• Function

Basic Molecular Biology4

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What to know about proteins?Structure

Chemical properties

Physical properties

Synthesis

How the sequence of a protein is determined?

Genetics

Function of the proteins

Structure/Function Relationship

Basic Molecular Biology 6

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The Central Dogma of Molecular Biology

Molecular biology seeks to explain the relationships between the structure and function of biological molecules and how these relationships contribute to the operation and control of biochemical processes.

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Applications of Molecular Biology

• Medicine• Agriculture• Pharmacy• Veterinary• Industry• Biological Sciences• etc

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• Medicine:BiochemistryParasitologyImmunologyBacteriologyVirologyPathologyLaboratory MedicineGeneticsetc.

Applications of Molecular Biology(Molecular Medicine)

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Tools used in molecular studies

• DNA cloning

DNA cloning facilitates the isolation and manipulation of fragments of an organism’s genome by replicating them independently as part of an autonomous vector.

Using Biological System

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• Polymerase Chain Reaction (PCR)

PCR is used to amplify a sequence of DNA using a pair of oligonucleotide primers each complementary to one end of the DNA target sequence.

These are extended towards each other by a thermostable DNA polymerase in a reaction cycle of three steps:

1. Denaturation

2. Primer annealing

3. Polymerization

Using Chemical System


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PCR MACHINES

• DNA Sequencing

The two main methods of DNA sequencing are the Maxam and Gilbert chemical method and Sanger’s enzymic method.

• RNA Sequencing• Protein Sequencing

DATA BASES

GenBank


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Some important events in the history of molecular biology

1871 Discovery of the DNA.

1943 DNA proves to be a genetic molecule capable of altering the heredity of bacteria :Avery, MacLeod and McCarty.

1953 Postulation of a complementary double-helical structure for DNA.

1960 Discovery of messenger RNA, and the demonstrationThat it carries the information that orders amino acids in proteins. 17

DNA: The Transforming Agent

In 1943, Oswald Avery, C. M. MacLeod, and M. McCarty on bacterium Streptococcus pneumoniae

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1961 Use of a synthetic messenger RNA molecule to work out the first letters of the genetic code.


1966 Establishment of the complete genetic code.

1970 Isolation of the first restriction enzyme, an enzyme that cuts DNA molecules at specific sites.

1973 Beginning of DNA cloning in E. coli.

1977 Formation of the first genetic engineering company(Genenetech) specifically found to use recombinant DNAmethods to make medically important drugs.

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1977 Development of procedures for the rapid sequencing of DNA.


1978 The Nobel Prize in Medicine was awarded for the discovery and use of restriction enzymes to Hamilton Smith and Daniel Nathans.

1978 Somatotatin becomes the first human hormone produced by using recombinant DNA.

1980 The Nobel Prize in Chemistry is awarded dually to for the formation of the first recombinant DNA molecules (Paul Berg) and the development of powerful methods for sequencing DNA to Gilbert and Sanger.to Gilbert and Sanger.

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1981 Sickle-cell anemia becomes the first genetic disease to be diagnosed antenatally directly at the gene level by restriction enzyme analysis of the DNA.


1982 Human insulin produced by recombinant DNA methods goes on the market under the trade name

Humulin.

1985 Use of heat stable DNA polymerase in PCR.

1990 First trial for gene therapy in humans

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THE CELL

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Cell Theory

Cells were discovered in 1665 by Robert Hooke.

Early studies of cells were conducted by

- Mathias Schleiden (1838)

- Theodor Schwann (1839)

Schleiden and Schwann proposed the Cell Theory.

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Cell Theory

Cell Theory

1. All organisms are composed of cells.

2. Cells are the smallest living things.

3. Cells arise only from pre-existing cells.

All cells today represent a continuous line of descent from the first living cells.

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Cell Theory

Cell size is limited.

-As cell size increases, it takes longer for material to diffuse from the cell membrane to the interior of the cell.

Surface area-to-volume ratio: as a cell increases in size, the volume increases 10x faster than the surface area

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Cell Theory

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Cell Theory

Microscopes are required to visualize cells.

Light microscopes can resolve structures that are 200nm apart.

Electron microscopes can resolve structures that are 0.2nm apart.

Visualizing Cells

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Cell Theory

All cells have certain structures in common.

1. genetic material – in a nucleoid or nucleus

2. cytoplasm – a semifluid matrix

3. plasma membrane – a phospholipid bilayer

Cell Characteristics

• Genetic material– single circular molecule of DNA in

prokaryotes– double helix located in nucleus in eukaryotes

– nuclear envelope (double membrane

• Cytoplasm fills cell interior – – sugars, amino acids, – proteins - organelles

• Plasma membrane encloses the cell phospholipid bilayer

Phospholipid

Membraneproteins 31

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Prokaryotic Cells

Prokaryotic cells lack a membrane-bound nucleus.

-genetic material is present in the nucleoid

Two types of prokaryotes:

-archaea

-bacteria

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Prokaryotic Cells

Prokaryotic cells possess

-genetic material in the nucleoid

-cytoplasm

-plasma membrane

-cell wall

-ribosomes

-no membrane-bound organelles

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Prokaryotic Cells

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Prokaryotic Cells

Prokaryotic cell walls-protect the cell and maintain cell shape

Bacterial cell walls-may be composed of peptidoglycan -may be Gram positive or Gram negative

Archaean cell walls lack peptidoglycan.

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Prokaryotic Cells

Flagella

-present in some prokaryotic cells

-used for locomotion

-rotary motion propels the cell

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Prokaryotic Cells

Generalized Eukaryotic Cell

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Eukaryotic Cells

Eukaryotic cells

-possess a membrane-bound nucleus

-are more complex than prokaryotic cells

-compartmentalize many cellular functions within organelles and the endomembrane system

-possess a cytoskeleton for support and to maintain cellular structure

The Plasma Membrane

B

A

D

C

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The Plasma Membrane

A. Phospholipid BilayerTwo layers of phospholipids

Hydrophilic head groups face

the water

Hydrophobic fatty acids face inside

Fatty acids may be saturated or unsaturated.

Fatty acid structure is adjusted to keep membranes fluid like oil, not solid like butter.

B. CholesterolAdds rigidity to the membrane.

Up to 30% of animal plasma membranes.41

The Plasma MembraneC. Proteins

Integral proteins span the membranePeripheral proteins are mostly on inside.Act as channels, transport, receptors, enzymesHelps to reinforce cell shape

D. Carbohydrates on the outsideGlycolipids—attached to phospholipidsGlycoproteins—attached to proteinsThe sugar chains form a sticky, sugar layer on the

outside of the plasma membrane called the glycocalyx.

Used in mammals for cell-cell identification as part of immune system, such as blood types.

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Eukaryotic Cells

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Eukaryotic Cells

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Eukaryotic Cells

Nucleus

-stores the genetic material of the cell in the form of multiple, linear chromosomes

-surrounded by a nuclear envelope composed of 2 phospholipid bilayers

-in chromosomes – DNA is organized with proteins to form chromatin

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Eukaryotic Cells

Chromosomes

• DNA of eukaryotes is divided into linear chromosomes. – exist as strands of chromatin, except during

cell division– associated with packaging histones,

packaging proteins• nucleosomes

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Eukaryotic Cells

Ribosomes

-the site of protein synthesis in the cell

-composed of ribosomal RNA and proteins

-found within the cytosol of the cytoplasm and attached to internal membranes

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Endomembrane System

Endomembrane system

-a series of membranes throughout the cytoplasm

-divides cell into compartments where different cellular functions occur

1. endoplasmic reticulum

2. Golgi apparatus

3. lysosomes

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Rough endoplasmic reticulum (RER)

-membranes that create a network of channels throughout the cytoplasm

-attachment of ribosomes to the membrane gives a rough appearance

-synthesis of proteins to be secreted, sent to lysosomes or plasma membrane

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Smooth endoplasmic reticulum (SER)

-relatively few ribosomes attached

-functions:

-synthesis of membrane lipids

-calcium storage

-detoxification of foreign substances

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Endomembrane System

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Golgi apparatus

-flattened stacks of interconnected membranes

-packaging and distribution of materials to different parts of the cell

-synthesis of cell wall components

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Lysosomes

-membrane bound vesicles containing digestive enzymes to break down macromolecules

-destroy cells or foreign matter that the cell has engulfed by phagocytosis

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Microbodies

-membrane bound vesicles

-contain enzymes

-not part of the endomembrane system

-glyoxysomes in plants contain enzymes for converting fats to carbohydrates

-peroxisomes contain oxidative enzymes and catalase

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Vacuoles-membrane-bound structures with various functions depending on the cell type

There are different types of vacuoles:-central vacuole in plant cells-contractile vacuole of some protists-vacuoles for storage

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Mitochondria

Mitochondrion

-organelles present in all types of eukaryotic cells

-contain oxidative metabolism enzymes for transferring the energy within macromolecules to ATP

-found in all types of eukaryotic cells

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Mitochondria

-surrounded by 2 membranes

-smooth outer membrane

-folded inner membrane with layers called cristae

-matrix is within the inner membrane

-intermembrane space is located between the two membranes

-contain their own DNA

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Mitochondria

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Cytoskeleton

-network of protein fibers found in all eukaryotic cells

-supports the shape of the cell

-keeps organelles in fixed locations

-helps move materials within the cell

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CytoskeletonCytoskeleton fibers include

-actin filaments – responsible for cellular contractions, crawling,

-microtubules – provide organization to the cell and move materials within the cell

-intermediate filaments – provide structural stability

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Cytoskeleton

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Cell Movement

Cell movement takes different forms.

-Crawling is accomplished via actin filaments and the protein myosin.

-Flagella undulate to move a cell.

-Cilia can be arranged in rows on the surface of a eukaryotic cell to propel a cell forward.

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Extracellular Structures

Extracellular structures include:

-cell walls of plants, fungi

-extracellular matrix surrounding animal cells

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Cell walls

-present surrounding the cells of plants, fungi, and some protists

-the carbohydrates present in the cell wall vary depending on the cell type:

-plant and protist cell walls - cellulose

-fungal cell walls - chitin

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Extracellular matrix (ECM)

-surrounds animal cells

-composed of glycoproteins and fibrous proteins such as collagen

-may be connected to the cytoplasm via integrin proteins present in the plasma membrane

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Extracellular Structures

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DIFFUSION AND OSMOSIS

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Diffusion

• Diffusion is the movement of molecules from an area of higher concentration to an area of lower concentration (which we call “down the concentration gradient”)

• Random movement of molecules fills the available space.

• Does not require cellular energy in the form of ATP.

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Factors Affecting Diffusion

Less diffusion More diffusion

Cooler Temperature Warmer

Larger Molecule size Smaller

Substrate State

SolidSolid Liquid Gas

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Osmosis and Tonicity

Osmosis is diffusion across a semi-permeable membrane.In cells, we usually compare water and salt concentrations inside & outside the cell.

Isotonic is salt concentrations same on both sides of the membrane

Hypotonic (“less salty”): one side is less salty than the other.

Hypertonic (“very salty”): one side is more salty than the other.

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Examples Using Red Blood Cells

Saltier outside than inside: water leaves cells, shriveling them.

Saltier inside than outside: water enters cells, swelling them, sometimes to bursting.

Salt conc. same inside and out. Normal for cells. No net movement of water.

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TRANSPORT ACROSS MEMBRANES

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Types of Transport Across Membranes

1. Passive Transport substances move down their concentration gradient

2. Facilitated Diffusion— protein-assisted diffusion

3. Active Transport— substances move against their concentration gradient; requires energy

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Passive Transport

• Diffusion across a membrane down the concentration gradient

• Does not require cellular energy (ATP)

• Usually pass through the lipid bilayer– Oxygen– Carbon dioxide– Small hydrocarbons (also hydrophobic)

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Facilitated TransportPassive transported assisted by proteins Goes down concentration gradient and so

does not require energy.For molecules that cannot easily pass

through lipid bilayer: ◦H2O◦ ions◦glucose, etc.

Osmosis with water occurs by facilitated transport using the protein aquaporin.

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Channel Proteins

Channel proteins are 1 of 2 types of integral membrane proteins in facilitated diffusion.◦Often made of 3 or more subunits (polypeptides)

grouped together.◦Have hydrophilic channel in the middle for

molecules to pass through.

◦Specific for type of molecule: H2O, Ca++, etc.

◦Many are gated and require a chemical or electrical stimulus to open the channel as in nerve cells (neurons).

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Examples of channel proteins

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Carrier Proteins

• Carrier proteins are facilitated transport proteins that bring molecules across the membrane by changing shape in a rocking motion.

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Active Transport

• Carries molecules against the concentration gradient

• Requires energy usually as ATP.

Ion pumps are active transport pumpsEstablish different concentrations of anions and cations across membrane so set up a charge across the membrane or membrane potential.

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The Sodium-Potassium Pump

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Technology

the cell