Introduction to Cell and Molecular

BiologyDr Helen E. Chick, PhD

Introduction video: https://www.youtube.com/watch?v=gFuEo2ccTPAhttp://www.promocell.com

Learning Objectives:• Knowledge and understanding in cell

and molecular biology research in life sciences and medicine

• Gain independent thought into research

• Encourage the new scientists of tomorrow’s world

Biology is fabulous!

Can you think of any cell and molecular biology words that come to mind?

• Write down key words Lets share them together.

Actin Filaments Animal cellsPlant cells

Plasma MembraneExtracellular Matrix

Golgi ApparatusLysosome mitosis DNA replicationCentrioles

Ribosomes Prokaryotes Eukaryotes Apoptosis

CentrosomesMitochondria

Rough Endoplasmic ReticulumGenoptype

Smooth Endoplasmic ReticulumNucleus

DNA methylationHistones epigeneticsCell Cycle (G0, G1, M and G2 phases) Genetic finger printing

Prophase, Metaphase, Anaphase and Telophase

There are some many………

Genetics, Organelles, Cells, Tissues and OrgansComplexity

We are all amazing because we are all made up of about 37 trillion cells of 200 different types.

• Cells are the fundamental unit of living organisms (the building blocks)

• The invention of the telescope made the Cosmos accessible to human observation - the microscope opened up smaller worlds.

• Cell biology has been studied since 16th century– Physiological properties– Structure– Organelles– Life cycle– Division – mitosis and meiosis– Cell death– Interactions between cells an their extracellular environment– Core for developmental biology, stem cell research, immunology and cancer biology

Cell Biology

Explore the cell: • Cell model• Cell video: https://www.youtube.com/watch?v=2KQbVr9kFO0What organelles can you stop in this video? – write them down.

Actin FilamentsPlasma Membrane

Extracellular MatrixGolgi Apparatus

LysosomeCentrioles

CentrosomesMitochondria

Rough Endoplasmic ReticulumSmooth Endoplasmic Reticulum

RibosomesNucleus

1595 – Jansen: developed the first light microscope1655 – Hooke: described ‘cells’ in cork.1833 – Brown: described the cell’s nucleus from the orchid.1839 – Schleiden & Schwann: proposed cell theory (all organisms are comprised of cells).1858 – Rudolf Virchow: omnis cellula e cellula - cells develop only from pre-existing cells by a process called cell division1894 – Altmann: first described mitochondria.1874 – Flemming: described chromosome behaviour during mitosis.1898 – Golgi: described the Golgi apparatus.1925 Gorter & Grendel: described the basic structure of the plasma membrane.1945 Porter et al. pioneers in this field of electron microscopy and were the first to identify the endoplasmic reticulum and many elements of the cytoskeleton. Cell biologist today are still researching the cell.

References: Mullock BM & Luzio JP, Theory of Organelle Biogenesis: a Historical Perspective (2005). Landes Biosciences, P. 1-13.

Muzzarello P, A Unifying Concept: the History of Cell Theory (1999). Nature Cell Biology, 1, E13-E15.

Exploring the History of Cell Biology:Timeline

• All known living things are made up of cells.• The cell is structural & functional unit of all living things.• All cells come from pre-existing cells by division (spontaneous

generation does not occur).• Cells contain hereditary information which is passed from cell

to cell during cell division.• Cells are similar in chemical composition.• All energy flow (metabolism & biochemistry) of life occurs

within cells.

References: Mullock BM & Luzio JP, Theory of Organelle Biogenesis: a Historical Perspective (2005). Landes Biosciences, P. 1-13.Muzzarello P, A Unifying Concept: the History of Cell Theory (1999). Nature Cell Biology, 1, E13-E15.

Modern Cell Theory

http://www.microbiologyonline.org.uk/media/transfer/doc/classifying_microbes_poster.pdf

The Society for General Microbiology

SimilaritiesProkaryotes/Bacteria Eukaryotes

• Plasma membrane (phospholipid bilayer and regulates permeability)• Genetic material – DNA• Cell Wall – except animal cells• Ribosome - catalyse protein synthesis• Cytoplasm/cytosol – comprising of water, glucose, proteins and ions.

The cell: https://www.youtube.com/watch?v=URUJD5NEXC8

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• Bacteria• Size: 1-10mm• Cell Wall (murein)• No distinct subcellular organelles• Circular chromosome – nucleoid• Often plasmids, RNA and Ribosomes• Unicellular or multicellular• Escherichia Coli (E. Coli) is most studied bacterium

• Plants, animals, fungi and protists (algae and protozoa)• Size: 10-100mm• Cell Wall – only plants, fungi and protists (cellulose)• Well defined subcellular compartments bounded by lipid

membranes• Cytoplasm consists of organelles, ribosomes, cytoskeleton

(shape, movement and organises many metabolic functions)

• Cytoskeleton: microtubules made of tublin & microfilaments made of actin.

• Most are multicellular• Differentiate to specialized tissue/cells

Learning resources from the society for general microbiologyPractical microbiology:http://www.microbiologyonline.org.uk/media/transfer/doc/sgm_basic_practical_microbiology_2.pdf

Looking at microbes: http://www.microbiologyonline.org.uk/students/microbe-passports-1#/home

http://www.abcam.com/alpha-smooth-muscle-actin-antibody-1a4-fitc-ab8211.html

Cytoskeleton – tublin and actin

Exploring organellesPr

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• Nucleus – Cellular DNA. Transcription & processing of RNA. Nuclear pores within the nuclear membrane.

• Mitochondria – Cellular respiration, the oxidation of nutrients to generate energy in the form of adenosine 5’-triphosphate (ATP). 1-2mm in diameter. 1000-2000 per cell. Smooth outer membrane & Inner folded membrane (cristae). Derived from prokaryotes and retain DNA (circular), RNA and protein machinery.

• Endoplasmic Reticulum (ER) – Cytoplasmic membrane system for lipid biosynthesis and xenobiotic metabolism. Smooth and Rough ER. Rough ER has ribosome attached for protein synthesis.

• Golgi Apparatus – Protein and lipids produced are packaged in the Golgi for final destination.

• Lysosomes – Small membrane-bound organelles & bud off from the Golgi. Consist of degradative enzymes for proteins, nucleic acid, lipids and carbohydrates (macromolecules).

• Centrioles – Regulator of the cell cycle and cytoskeletal organisation.

• Nucleoid – composed of circular double-stranded DNA.

• Plasmid DNA – Short circular DNA and replicates independently of the cell genome.

• Mesosome – Folds of the plasma membrane with associated respiration enzymes. Instead of mitochondria.

• Ribosomes – Smaller, scattered throughout the cytoplasm

• Pilli – protein rods for cell-cell attachment and DNA transfer.

• Flagellum – Motility of many bacteria• Cell Wall – Rigid and made up of murein

(polysaccharide cross-linked by peptide chains). Gram-positive thicker walls compared to Gram-negative. Protection from lysozymes and penicillin.

• Capsule – slime layer of mucilage and helps bacteria form colonies.

Explore molecular biology and genetics: • What is this model demonstrating?• Genome video: https://

www.youtube.com/watch?v=VJycRYBNtwY• What Molecular and Genetic words/phrases can you stop in this

video? – write them down.

Molecular Biology

Cells archive information in the form of DNA, which serves as a master set of instructions for building proteins.

Protein functions: organelle biosynthesis, structural supports, catalysts of biochemical reactions and aid in the cell's internal and external environments/signalling.

The length of DNA in each cell is 2 metres long.

Discuss: What Molecular & Genetic words/phrases can you spot in this video?

human genome projectCells mitochondrial DNA

Haplotype Nucleus23 pairs of chromosomes & 46 chromosome

DNAPhenotype Genetics

2 metres long DNAGenetic coding (4 bases)

RNA polymerase SNPtranscription of DNA in the nucleus to mRNA

RibosomesGenetic Variants tRNA Tandem

RepeatsAmino Acids

Polypeptide Mutations GenotypeTranslation of mRNA to proteins in the cytoplasm

Human complexity – muscle, hair, eye colour, hormones, biochemical reactions, diseases etc..........

Exploring the History of Molecular Biology and Genetics Timelines to explore: http://www.bioinformatics.nl/webportal/background/timeline.htmlhttp://www.dnai.org/timeline/

February 28th 1952 – The double HelixThis is the date when Watson & Crick (at the University of Cambridge) announced the structure of DNA in the Eagle pub, Cambridge

Molecular Biology and genetics

• DNA: DeoxyriboNucleic Acid RNA: RiboNucleic Acid• Bases: Adenine (A) and Guanine (G) = Purines

Cytosine (C) & Thymine (T) = Pyrimidines• RNA has Uracil instead of Thymine• Base sugar = Nucleoside (Deoxyribonucleoside & Ribonucleoside)• Base sugar and phosphate unit = nucleotide . Many form a polynucleotide• Phosphodiester bonds = link nucleotides for nucleic acid polymer• Hydrogen bonding between paired bases• Each chain is complimentary

Pentose sugarRiboseDeoxyribose

Phosphate group

Phosphodiester bond

DNA PackagingDNA replication

DNA Double Helix

Minor GrooveMajor Groove

RNA• DNA transcribed into mRNA by RNA polymerase• RNA used as a primer in DNA replication• Ribosomal RNA• tRNA aids in translation of mRNA into protein

• Chromatin – DNA bound to histones (DNA packaging proteins)

• DNA and histones are organised into nucleosomes. Eight histones = histone octamer

• These are linked by linker DNA

1. Double helix is unwound and the base-pairs are separated by DNA helicase.

2. Semi-conservative replication3. Nucleotides are linked covalently by DNA polymerase4. Lagging and leading strands5. DNA ligase for the shorts strands on the lagging strand

Molecular Biology and genetics

Transcription & translation

Molecular Biology and geneticsPolymerase Chain Reaction (PCR): DNA

amplification

Denaturation at 94oC

Primer annealing at 55oC

Polymerization 72oC by DNA polymerase

Applications:• Diagnosis• Therapy• Historical studies• Forensic analysis• Cloning and sequencing • Research – genome, genetic variants• Maybe Dinosaur production in the

future – Jurassic world!

Molecular Biology and genetics

Figure 2: The polymerase chain reaction (PCR). Invented in the 1980sTwo key innovations facilitated the use of PCR in the laboratory: the discovery of a DNA polymerase that is stable at the high temperatures used in step 1 of PCR and the development of automated thermal cyclers (machines that bring about the rapid temperature changes necessary for the different steps of PCR).© 2014 Nature Education Adapted from Pierce, Benjamin. Genetics: A Conceptual Approach, 2nd ed. All rights reserved.

PCR is like a photocopier for DNA

DNA electrophoresisApplications:• DNA finger

printing for Parentage and Forensics

• Genetic cloning – DNA profiling

https://www.thenakedscientists.com/podcasts/science-scrapbook/how-does-dna-fingerprinting-work

Genetic finger printing video:

• Mitosis – Cell division https://www.labtube.tv/video/mesc-cell-mitosis

• Apoptosis – programmed cell death:https://www.labtube.tv/video/apoptosis

• Necrosis - premature death of cells due to injury, infection, cancer, infarction, toxins and inflammation.

• Migration, differentiation etc…

Molecular Biology and geneticsEpigenetics:• Genetic control • Switch genes on or off and determine which proteins are

transcribed: DNA methylation Histone modifications Nucleosome positioning

• All critical for regulating gene expression.• Our cells all have the same DNA, but our bodies contain many

different types of cells: neurons, liver cells, pancreatic cells, inflammatory cells, and others.

• Because certain sets of genes that are "turned on" or expressed, as well as other sets that are "turned off" or inhibited.

• Differential expression.

Reference: http://www.nature.com/scitable/topicpage/epigenetic-influences-and-disease-895#

Restriction enzymes and DNA modifying enzymes (i.e. genetic editing) For example, EcoRI restriction enzyme was once a defend mechanism to remove foreign DNA in bacteria, but we have genetically engineered them to be useful tools in cloning – NEB (Don Comb).

Founded in the mid-1970s as a collective of scientists committed to developing innovative products for the life sciences industry, New England Biolabs is now a recognized world leader in the discovery, development and commercialization of recombinant and native enzymes for genomic research.

https://www.neb.com/about-neb

PCR, gene expression, cellular analysis, RNA analysis and epigenetics

Genetic editing tools:• Over 3 billion base pairs in a

single human’s DNA• It is difficult to pinpoint what

sequences are important in health and disease

• Cas9/CRISPR are like scissors for precise gene targeting

Chromatin / Epigenetics Resources Protein Acetylation Histone Methylation Examples of Crosstalk Between Post-translational Modifications

MAP Kinase Signaling Resources MAPK/Erk in Growth and Differentiation G-Protein-Coupled Receptors Signaling to MAPK/Erk SAPK/JNK Signaling Cascades p38 MAPK Signaling Pathways

Apoptosis Resources Regulation of Apoptosis Inhibition of Apoptosis Death Receptor Signaling Mitochondrial Control of Apoptosis

Autophagy Resources Autophagy Signaling

PI3K / Akt Signaling Resources PI3K / Akt Signaling mTOR Signaling

Translational Control, Protein Synthesis, and RNA Regulation Resources Regulation of eIF4E and p70 S6 Kinase Regulation of eIF2 Overview: Translational Control

Calcium, cAMP and Lipid Signaling Resources Protein Kinase C Signaling Phospholipase Signaling

Cell Cycle, Checkpoint Control and DNA Damage Resources G1/S Checkpoint G2M/DNA Damage Checkpoint

Cellular Metabolism Resources Insulin Receptor Signaling AMPK Signaling Warburg Effect Hypoxia Signaling

Stem Cell Markers, Development and Differentiation Hippo Signaling Pathway ESC Pluripotency and Differentiation Pathway Stem Cell & Lineage Markers Wnt / β-Catenin Signaling Pathway Notch Signaling Pathway Hedgehog Signaling Pathway TGF-β Signaling Pathway

Immunology and Inflammation Jak/Stat: IL-6 Receptor Signaling NF-κB Signaling Toll-Like Receptor Signaling B Cell Receptor Signaling T-Cell Receptor Signaling Inflammasome Signaling

Tyrosine Kinase Resources ErbB / HER Signaling

Angiogenesis Resources Angiogenesis

Adhesion and Extracellular Matrix Resources Adherens Junction Dynamics

Neuroscience Resources Amyloid Plaque and Neurofibrillary Tangle Formation in Alzheimer's Disease Dopamine Signaling in Parkinson's Disease Vesicle Trafficking Presynaptic Signaling

Cytoskeletal Regulation and Vesicle Trafficking Resources Regulation of Microtubule Dynamics Regulation of Actin Dynamics

Nuclear Receptor Resources Nuclear Receptor Signaling

Ubiquitin and Ubiquitin-Like Protein Resources Ubiquitin / Proteasome

Protein FoldingOrganelle Markers

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Cell signalling pathways

http://www.cellsignal.com/contents/science/cst-pathways/science-pathways

Cell signalling:

Explanation to cell and molecular biology research

Organelle Diseases:Mitochondrial

diseases• Dysfunctional

mitochondria• Mutations in

mitochondrial DNA and nuclear DNA

• 1 in 200 children in the UK

• Mitochondrial DNA disease is passed down from mother to child

• Currently no cure• Unlikely to survive

childhood• The Wellcome Trust

Centre for Mitochondrial Research at Newcastle University has developed a treatment known as mitochondrial donation (http://www.wellcome.ac.uk/About-us/Policy/Spotlight-issues/Mitochondrial-diseases/)

Epigenetics related diseases/disorders• Cancer• Mental Retardation• Obesity• Immunodeficiency(http://www.nature.com/scitable/topicpage/epigenetic-influences-and-disease-895)(http://www.ncbi.nlm.nih.gov/pubmed/20944598)

Cancer• Epigenetics• DNA mutations• Inherited genes• Cell cycle• Hormones and

growth factors – proliferation, differentiation and apoptosis of cells

http://www.nature.com/scitable/search-scitable?criteria=cancer

Biotechnology and Biopharmaceutical• Gene and Cell Therapy• Antibodies for therapy and

diagnostics• Gene engineering – e.g

Insulin, cloning• Stem cells – iPSC• Personalised medicine• DiagnosisCovered in next lecture.

Metabolic Diseases – for example Diabetes(http://www.nlm.nih.gov/medlineplus/metabolicdisorders.html)

Cell cycle related diseases/disorders:• Cancer• Cardiovascular diseases• Infection• Inflammation• Neurodegenerative(http://www.ncbi.nlm.nih.gov/pubmed/20964732)

Cell signalling diseases/disorders– for example CVD, tissue repair, neurological

The explosion of knowledge brought about by improvements in microscopy, biochemistry, and genetics has led to a depth of

understanding of cell structure and function undreamed of by the earliest cell biologists.

PublicityNature (http://www.nature.com/)

New Scientist (http://www.newscientist.com/)

Naked Scientist (http://www.thenakedscientists.com/)

LabTube (https://www.labtube.tv/)

Search engines for journalsPubmed (http://www.ncbi.nlm.nih.gov/pubmed)

Google Scholar (https://scholar.google.co.uk/)

Biotechology websitesLife Technologies

(https://www.lifetechnologies.com/uk/en/home.html) Abcam

http://www.abcam.com/

New England Biolabs (NEB) (http://www.neb.uk.com/)

Illumina (http://www.illumina.com/)

Horizon (http://www.horizondiscovery.com/ )

Company websitesGSK (http://uk.gsk.com/)

Medimmune (https://www.medimmune.com/)

Babraham Research park (http://www.babraham.co.uk/about/about.html)

Pfizer (http://www.pfizer.co.uk/)

OxfordBiomedica (http://www.oxfordbiomedica.co.uk/)

How to research the cellular and molecular universes yourself:

Institute websitesWellcome Trust Sanger Institute (https://www.sanger.ac.uk/)

Ensembl (http://www.ensembl.org/index.html) Genome databases for vertebrates and other eukaryotic species.

Human Genome (https://www.genome.gov/11006943)

British Society of Gene and Cell Therapy (http://www.bsgct.org/en/)

European Society of Gene and Cell Therapy(http://www.esgct.eu/)

American Society of Gene and Cell Therapy (http://www.asgct.org/)

‘UNIVERSITY CHALLENGE’ QUIZ

• Form groups – four teams required• Are you ready for the cell and molecule biology quiz?!

References• Bianconi E. et al. An estimation of the number of cells in the human

body (2013). Ann Hum Biol; 40 (6): 463-71.• W. R. Pickering. Oxford Revision guides, AS & A level Human Biology

through diagrams.• Turner P. et al. Bios Instant Notes, Molecular Biology, 3rd edition. • All images are from Clip Art on MicroSoft Word

Question two: Name these organelles (1 point for each organelle)?

Simple DNA extraction

Alkaline cell lysis to break open the cells and organelles

Alcohol/ethanol precipitation: DNA remaining in the aqueous layer is concentrated by ethanol precipitation.