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MCB REVISION
LECTURE 1 & 2- RNA SYNTHESIS
General info: C value paradox- C= amount of DNA in one set of chromosomes
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Satellite DNA accounts for the extra amount of DNA found in less complex organisms
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RNA synthesis: DNA -> Transcription -> RNA transcript= serves directly as mRNA [Pro], processed to become mRNA [Euk] -> Translation -> Peptide
Prokaryote
Initiation: RNA polymerase [2α & 2β subunit] binds to the Promoter region of DNA via σ-factors at -10 & -35 box
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Once bound tightly, RNA polymerase II is a closed complex, it unwinds a short section of DNA forming a open-complex
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When enough nucleotides have been added along the 3’ end, the Sigma subunit [σ] dissociates
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Termination: [Rho independent/Intrinsic termination]: Rho protein not required
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The Inverted repeats are complementary sequences in reverse on DNA
When the core enzyme has transcripted these sequences, the strand bends and the inverted sequences H bond
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[Rho dependant]: Rho factor [protein] travels along RNA transcript until it reaches RNA polymerase
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Eukaroytes: Many control points- DNA replication
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–RNA processing
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–Post-translational processing
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Overcomes background expression of endogenous genes
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–Promoter= activation of the gene
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This induces expression of the particular protein of interest
->Transgenic: foreign gene inserted into fertilised egg, grows and is incorporated into genes [reporter genes]
Cell transformation strategy: Microinjections= Uses glass pipette [0
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–Used on cells without a wall
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DNA integrates into chromosome [randomly]
Calcium= Calcium precipitate used to add DNA into cells
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Low transformation efficiency
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–High transformation efficiency
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Gene guns= DNA is coated with Tungsten beads [metal]
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–Efficient
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–The Lipids then carry the DNA through the cell membrane
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Viral vectors= Uses a virus for delivery but exploiting the virus’ features
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[Retrovirus= encapsulated virus with 2 singled stranded copies of RNA genome]
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–Safety precautions must be taken when handling the virus
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–Kills cells which do not contain NeoR gene
Co-transforms cells with DNA of interest and NeoR gene
Eurkayotes- RNA synthesis; Initiation= TATA box located in DNA strand
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After binding event, TFIID recruits RNA polymerase II along with transcription factors TFII A,B,E,F,H
Combination of all these TFs & RNA polymerase II is known as the ‘Pre-initiation complex’/Basal complex
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–Then TFII H,E,B dissociate
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Then, RNA polymerase II CTD [c terminus domain] becomes phosphorylated
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TFII A & D remain at the TATA box, RNA polymerase II & TFII F move down the DNA and synthesis mRNA
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Termination= Ribonuclease then cleaves the mRNA strand
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-5’ Cap is a methylated gene added to RNA degradation
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–Introns discovered by R loop experiment
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Nuclei contains snRNA [small nuclear RNA], which associate to splicing enzymes forming SNURPs
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–They bind to mRNA and create spliceosomes which remove introns
-Poll II promoters: TATA box is unique to RNA poll II, the promoter is double stranded
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-PolyA= addition site where transcription stops [downstream flanking region], comes after stop codon
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–mRNA that has not had its introns removed is known as Precursor mRNA
[Cis] Enhancer region: Retains function even when gene of interest is moved far or reversed
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-Cis acting elements involve DNA sequences on the same chromosome as the gene
[Trans-acting element] Gene products interact with cis-acting elements
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Polymerase I: Only transcribes genes for rRNA [ribosomal] from a single type of Promoter
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–Transcription factors bind to enhancers
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Polymerase III: Transcribes DNA to synthesise 5s rRNA, tRNA and small RNAs
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LECTURE 3- RECOMBINANT DNA TECHNOLOGY
Gene cloning: Replicating copies of a gene for downstream applications: sequencing, mutagenesis, genotyping/ heterologous protein expression
Cell based cloning: Insertion of genetically modified material into a cell for the purpose of it to replicate and pass on the newly introduced
genetic material into progeny cells
Vector: A DNA molecule which is used as an artificial vehicle to transfer foreign genetic material from one organism to another
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–Types= Plasmid, Viral, Cosmid, artificial chromosomes
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–Usually done using a vector
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Restriction; asymmetrical cutting [sticky-cohesive overhanging ends]
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–Diagnostic tool
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–Research
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-3 Million Bp
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-6,000 genes with known function
LECTURE 4- RDT 2
DNA analysis- Gel electrophoresis: Gel staining [Ethidium bromide-Intercalating agent]
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–Separation also according to Conformation [shape]
->Plasmids can be supercoiled [further] or open circle
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–Calibration graph used to work out unknown length
Human DNA- 3 x 109 bp
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Identifying particular band/(s): Transfer genetic material from the gel to a membrane- Use a probe to identify the sequence & Southern blot
-Northern Blot: Study gene expression by detecting isolated RNA
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->Denature the gel with NaOH
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->Capillary blot- absorbent paper at the top, gel at the bottom
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–Identifying related family members
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–Gene conservation across species
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-Eastern Blot: Analyse protein-Post translational modifications
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–Single stranded DNA/RNA
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Transgenic mice became x7 more active than control mice
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–cDNA better since it is derived from mRNA which contains the complete coding part of DNA
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-34 mice were microinjected, 6 where [+] transgenic mice
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–DNA isolated from their tails, digested with PstI
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–Nylon hybridisation membrane extracts the DNA and the probe screens it
LECTURE 5- RDT 3
Southern Blot: Can detect SNPs [single nucleotide polymorphisms] that alter the DNA restriction sites
-SNPs: Single base pair changes
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–Majority occur in the Non-coding region of DNA
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g
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–GAG changed to GTG, this alters Glu to Val at position 6
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=>Probe: alkaline phosphatase-oligonucleotide conjugate
-Other techniques: Western blot= SDS polyacrylamide gel electrophoresis -> Protein blot on nitrocellulose -> Label with specific antibody
-Alkaline Phosphatase: Removes a 5’Phosphate group from the DNA or RNA
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–>Removing 5’P from DNA fragments before labelling with radioactive phosphate
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-RT-PCR: Cell/tissue -> RNA is isolated from cell
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-> cDNA is synthesised
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-PEPCK-C determination: Mouse tissues were homogenized
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LECTURE 6- ENZYMES IN RDT
Restriction endonuclease
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–Polynucleotide Kinase
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–Terminal transferase
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–Nuclease
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->When ALP is added, the phosphates from the ends of the DNA/RNA strands are removed
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->If removed, Ligation of the strands are prevented
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-Terminal transferase: Template independent polymerase enzyme which catalyses addition of deoxynucleotides to 3’ end of DNA
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e
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–Enzyme is isolated and purified from retroviruses
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–S1 nuclease digestion removes unhybridised RNA/DNA probes [nuclease protection assay]
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Klenow fragment of DNA pol I [large protein fragment made via cleavage of DNA pol I from e
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(I) Comes from e
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–Makes DNA in 5’-3’
DNA labelling: Techniques= Nick Translation
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-3’ or 5’ end labelling
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–Deoxy/Dideoxynucleotides
Non-radioactive methods= DIG
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–Biotin
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]
-Nick translation: Requires simultaneous action 2 two enzymes
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–In Mg2+ presence, DNase I becomes a Single stranded endonuclease
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coli]: Adds labelled nucleotides to the 3’ ends created by DNase I during cleaving
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Klenow fragments then polymerise extending the oligodnucleotides with labelled dNTP+s
-Non radioactive/ECL: Double stranded DNA is denatured with boiling water
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-Flourescent probe: DNA probe is synthesised using modified dNTPs and Flourochrome side group
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-DNA microarrays: mRNA extracted to determine the gene expression
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Involves hybridisation of nucleic acids to oligonucleotide probes [attached to solid support]
->Molecular analysis of DNA: Bioinformatics
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–Restriction mapping
-DNA sequencing methods: Manual- Using ssDNA template [Sanger]
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–Elongation via DNA Pol [Klenow fragment]
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–PCR
LECTURE 7- MIGHTY MOUSE, VECTORS AND CLONING
DNA sequencing- the next step: Bioinformatics= assigns the DNA sequence to gene function
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–Disease mapping
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->Mighty Mouse: PEPCK-C, a crucial gluconeogenic enzyme which catalyses diversion of TCA intermediates towards gluconeogenesis
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–Western blot used in overexpression studies
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->Skeletal muscles were targeted for over expression of PEPCK_C as they do not synthesise their own Glucose
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–Case study= PKC/HINT1 knockout mice
->PCK/HINT1 gene: Protein Kinase C interacting protein [small]
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]
Absence of this gene is associated with Mood dysfunction behaviour, depression and anxiety
=>Hypothesis testing: Create a PCK/HINT1 knockout mouse [lacking gene] and compare to the wild type
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-Recombinant DNA cloning: DNA fragment for cloning enzymatically inserted into a Plasmid vector
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coli cells with the plasmid vector in CaCl2 presence & culture on nutrient agar containing Ampicillin
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Cells replicate the plasmid and begin multiplying
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->Choice of Vectors: Features- multiple cloning sites, a replication of origin, and selectable markers [i
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Amp gene]- easier to grow and detect
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–Viral [Lambda vector]
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e
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Artificial chromosomes
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–β Galactosidase
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-> White colonies
β Gal + Xgal [colourless] -> Blue colonies
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Can be replicated as a plasmid, and/or packaged into a single stranded DNA viral particle
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Used in various techniques; Electroporation
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–BACs [bacterial]
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–PACs [P1 viral]
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–Very large inserts [500kb-1mb]
Contain Telomeres [stabilise chromosome ends], Centromeric genes [partition stability], Ori of replication and Ampicillin selective marker
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-BACs: Similar to E
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–Can accommodate DNA fragments up to 300kb
Contain: Ori of replication
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–Derived from F-factor
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Advantages over YACs: More stable
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–Speed of growth of e
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–Simpler to purify
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->Capable of propagating large complex DNA inserts within E
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e
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TB
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–Vector= Cosmid [max 40kb therefore 2500 cosmids needed
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-EST [Expressed sequence tag]: A small bit of sequence representing gene of interest
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e
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Expression vectors must be able to perform DNA transcription to form mRNA and then Translation
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–Phage [λgt11]
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->Adding a Tag: A tag could already be attached to the vector which it would then bind to the c/n terminus of the DNA
Second way is to build the tag, design a primer with the tag connected and anneal it with the DNA in PCR
->Products produced: TPA [prevents blood clots]
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–Tissue growth factor β [wound healing]
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Reporter Genes: LacZ gene -> Forms β-gal -> Asssay=Histochemical
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-Homogolous recombination places the target gene with a reporter gene, usually located next to a promoter
Protein-Protein interactions: Said to be involved in Two-hybrid systems i
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What yeast can do
Peroxins: Human proteins encoded by PEX gene
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LECTURE 9- TRANSFORMATION AND TRANSGENESIS
Transgenic plants: Improving crop properties by Specific plant breeding and Inhibition of deleterious effects of insects/weeds with chemicalsides
-Introducing DNA into plant: Gene gun
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->Methods: Dicot transformation [soybean/squash/tomato], using Agrobacterium-mediated conjugation
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e
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-First GMO in the market= Flavr Savr Tomato
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->Nutritional enhancement: Golden rice- genetically modified to produce β-Carotene [Vitamin A precursor], VitA deficiency cause blindness
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–Transcription factors DEL & ROS1 in snapdragon tomato; causes Anthrocyanin accumulation [protection]
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–Transgenes were identified with Southern blot & PCR analysis
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e
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Virus interacts with mammalian cells in 2 ways
Lytic pathway: Within permissive cells which lead to production of virus particles in the cell and causes death
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-Permissive refers to the fact that viruses can circumvent host cell defences and replicate
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–Results from differential splicing of a single transcript
->Transformed SV40 Cos-1: Derived from Simian Kidney CV-1 cells
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–Expression constructs with SV40 promoter is introduced into these COS cells, the vector is replicated highly by T Ag
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-Creating Transgenic mice: Injecting cloned genes into nucleus of a fertilised egg
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->Producing Transgenic mice carrying hGx: Human growth hormonal structural gene
-Assessing expression of Transgene: RNA analysis- Total RNA extracted from the mice tissue via QuickPep total RNA & Northern Blotting
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–Libraries are collections of cloned fragments which contain 1 copy of every sequence in the entire genome
-Genomic equivalent: Number of clones in a perfect library
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–Insert DNA fragments into a vector forming Recombinant DNA
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–Genomic library formed
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coli with phage
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–Contains only the information from the gene’s exons
-Construction: Prepare total mRNA from tissue -> Add Reverse transcriptase + 4 dNTPs + Primers for cDNA synthesis [RT-PCR]
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2nd Strand cDNA synthesised with +dXTPs
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Genomic & cDNA library comparison: Every tissue in the body of a multicellular organism can produce the same genomic library
DNA fragments in the genomic library collectively carries the DNA of the entire genome
However, every tissue in the body produces a different cDNA library
Clones in the library represent the genes being transcribed hence active coding regions ‘exons’
Identifying Clones: Hybridisation- identifies similar DNA sequences
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Transfer clones onto a Nylon membrane
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-Screen the library: Expose the probes to the clones on membrane
Autoradiography/fluorescence determines matching clones
Stringency: Temperature [high temp= high stringency]
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–GC richness
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–Similarity to target [Factors]
-Single stranded nucleic acids Hydrogen bond to each other efficiently [20-25 degrees below their melting point]
->Stringency conditions: Temp [high], Salt, Denaturing additives [formaldehyde]
Probes: Nuclei acid probes
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–Expression cDNA libraries can be screened using specific antibody probes to identify
recombinant clones expressing fusion proteins
->General technique for probe labelling; Spread Library onto petri dish -> Overlay plate with Nitrocellulose/ nylon membrane to pick cells up
Lyse cells and denature with NaOH -> Bake and treat with UV light to bind DNA strands
Identify the fluorescent spots
->Locating specific clone in DNA library summary: Screen DNA & genomic libraries
Probes- Heterologous probes [specific DNA sequences in library]
Oligonucleotide probes [identify genes or cDNA]
Expression screening
Complementation of mutations- gene identification [fixing phenotype from mutant]
LECTURE 11- HYBRIDISATION
DNA microarrays: Arrays are physical collections of DNA sequences
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–PCR generated cDNA; EST
Sequences are spotted onto solid surfaces [glass or nylon] via robots
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–Probes are used to determine complementary binding
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->Types: Microspotting- Delivers the DNA clone Microarrays, printing them on a microscopic slide
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–Determination of expression level of genes
=>Preparation: Hybridisation- Prepare cDNA probes from mRNA isolated from control and treated cells
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Hybridise both probes to one microarray
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-Gene discovery
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–Drug discovery [pharmacogenomics]
-Toxicology research [toxicogenomics]: correlations bet
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–Cytochrome P450 polymorphism

LECTURE 12-13: PROTEIN STRUCTURE CONCEPTS
Hydrophilic: Polar ‘water soluble’
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–Contain extra COO- making it acidic
Lys, Arg & His= Basic & Polar
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–No charged side groups
Gly= Exception, Has a Hydrogen as a R group
Proteins composed of domains: A single polypeptide chain can fold into 2 or more compact spatially distinct regions [Domains]
-Domains: Formed from one chain
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-Domain shuffling: Domains appear in more than one protein
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Electrophoresis: Electric force [fe = Eq]
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–q= molecule which charge
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–η= medium viscosity
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–v= molecule viscosity
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–Velocity is proportional to q/r [charge density]
-Gel sieiving effect: The more acrylamide gel used, the smaller the pores will be
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Velocity of charged proteins moving through the gel in an electric field depends on: Protein charge density
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–Shape
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–Enzyme separation
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SDS [sodium dodecyl sulfate] PAGE: SDS included in protein sample, gel and running buffer
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-Gives proteins high [-] charge, masks proteins own charge
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-Mercaptoethanol: Used in SDS, included in protein sample- Breaks cysteine Disulphide bonds
Unfolds protein structure making all proteins the same conformation
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–Smaller proteins migrate further
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–Silver staining [x100 more sensitive than Coomaisse]
->Protein mass can be determined: Mobility of proteins is Inversely proportional to LogMass
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Isoelectric Focusing [IEF]: Electrophoretic separation of proteins according to their respective Isoelectric Points
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–Depends on the loss or gain of hydrogen at NH2 or COOH ends
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IEF mechanism: Low pH end connected to [+] electrode
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Proteins will then migrate until the pH of the gel is equal to its Isoelectric point
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LECTURE 13-14- CONTINUATION OF ELECTROPHORETIC TECHNIQUES
Proteins net charge alter with pH
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-Proteins with charge will only migrate, once the pH is reached where the protein loses its charge the protein will stop migrating: Isoelectric point
->Protein X has a pI of 6
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0
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0? Protein X
->Glu6-Val mutation in haemoglobin associated with sickle cell anaemia: Increases pI
=>Mutant form loses Glu [- charged], changed to Val [no charge]
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2D PAGE: Uses Isolectric focusing [IEF] to separate proteins according to Isoelectric points
IEF strip is placed onto SDS PAGE gel
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IEF must be performed first as SDS binds to the proteins altering their charge to 0 allowing no charge separation to occur
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-> Incubate membrane with Primary antibodies -> Incubate with 2nd Ab ->
Detect emitted light form bound antibody
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–Highly specific antibody-antigen interaction
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-Beads usually used in the columns
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–Agarose
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–Cellulose
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–Porous glass/silica
Gel filtration chromatography= Mixture of Proteins added to the column -> Smaller proteins move through pores of beads hence are slower
Larger proteins move through column quicker as they move between the beads therefore emerge first
Ion exchange chromatography= Proteins are separated according to Net electrical charge [total charge]
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-Bead + CM [-]
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–Positive proteins will stick to the beads, negative proteins will elute through
Elution with buffer of different pH or higher ionic strength increases effectiveness of elution
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->Beads covalently linked to ligands of the proteins which are targeted
=> Mixture of target protein and contaminants added to column -> Target protein binds to ligand on beads filtering it from sample
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–Promotes hydrophobic interactions
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–Alkyl side groups makes very hydrophobic proteins interact
HPLC: Enhanced version of other methods
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High pressure is used- increases flow rate through the fine matrix [stainless steel columns]

-Protein and its mutant separates in Reverse phase chromatography but not in ion exchange chromatography
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–If proteins are not separated in Ion exchange, then mutation has not altered
charge
LECTURE 15-16: PROTEOMICS
Proteome: A set of proteins expressed in a particular cell at a particular time in a set of specific conditions [differentiation, disease, drugs]
-Single gene can produce many proteins via Alternative splicing and Post-translational modifications [phosphorylation]
->Average: 1 gene= 40 proteins
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=>2D PAGE: Identifies proteome components
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Proteomic analysis: Comparison between a diseased person and health person with 2D PAGE pattern allows disease biomarker identification
-Cancer biomarkers found via Proteomics
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–Mass spec
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->Excess PTC washed with benzene
Cleavage- Dried PTC is treated with anhydrous acid cleaving the molecule
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–Then identified via HPLC using UV absorbance
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==>Some proteins require cleavage before sequencing- Edman degradation requires free amino groups at the N terminal
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–Enzymatic cleavage: Trypsin
-Protein Microarray: Microscope slide with microscopic spots each containing a type of protein
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Proteins are ionised
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–Acceleration depends on mass & charge of protein
->Proteins must be digested first into peptides to remove complications of Post-translational modifications and hydrophobicity
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->MALDI/ ESI is used to ionise proteins and peptides to prevent fragmentation
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Energy is absorbed into the Matrix -> Transferred to the protein becoming Ionised -> Protein enters the gas phase
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=->Mass determination: M/Z -> peak at 5700 with a charge of +1
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–Droplets contain sample + solvent with charges (H+)
Droplets begin to slowly lose solvent molecules in mixture giving sample ions with multiple charges
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LECTURE 16-17: PROTEIN STRUCTURE & FUNCTION OF SERINE PROTEASES
Proteolysis= Hydrolysis of peptide bonds in proteins
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–Split into 2 domains
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–Cleavage of signalling sequences after the protein has reached its destination
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Conversion of precursor proteins into their final form [Proinsulin -> Insulin]
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–Both have Alpha helices & Beta sheets
Chymotryspin: Digests bonds after aromatic residues [Trp, Phe, Tyr] & those with long hydrophobic chains [Met, Leu]
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–C=O-N- is cut
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–Serine proteases are made like this [Trypsinogen -> Trypsin, Plasminogen -> Plasmin]
Stored in Zymogen granules within the Pancreas and only secreted into SI and are then activate
Proteases are required to cleave Zymogens to form active Proteases
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–Inhibitors required to stop Proteolysis
Inhibitor: Pancreatic Trypsin inhibitor- binds strongly to the active site of Trypsin
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–Low/defective AAT binding means excessive Elastase in the body
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Not enough Elastin to help Alveoli during expiration, air is trapped meaning less O2 in the blood
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Hereditary- Z mutation of Glu342 -> Lys, slows AAT secretion
-Chymotrypsin activation: Chymotrypsinogen [inactive] –[Trypsin]---> π-Chymotrypsin [active] --> α-Chymotrypsin [active]
The result of cleavage causes conformational change forming specific active sites for aromatic & long hydrophobics
Serine: Contains a Catalytic triad- Ser195, His57, Asp102 form the active site
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A substrate binding to Serine will have the aromatic ring attached to the binding pocket, and the Cleavage site positioned above Ser-195
Ser-195 transfers H+ to His-57 via help of Asp102
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H+ is transferred from His57 to the substrate
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Water molecule enters the active site and H bonds with His57
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H+ transferred back from His57 to Ser195 & N terminal peptide is released
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–High solubility
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Fibrous Proteins: Rod like shape
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–Structure i
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Tropomyosin, Myosin, Elastin, Fibroin, Collagen, Keratin
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–Located in Bones, Teeth, Cornea, Skin and Connective tissue
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–Sequence: Tripeptide repeat of Gly-Pro-HydroxyPro [very common]
...
Stabilised by steric ring repulsion between Pro & HydroyxyPRo
...

=>Collagen molecules are Trimers [triple helix shape]
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=>Hydrogen bonds form between the chains of the Triple helix [NH, CO & OH groups]
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-Type 1 Collagen: 90% of all collagen- Triple Helices forms Collagen fibirls- stabilised by lysine and hydrolysine cross-linkage
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–Non-helical provides flexibility
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–Bleeding gums, Loss of teeth
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Vitamin C required for Prolyl Hydroylase activity
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-Keratin: Dimer molecules
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Cross-linkage occurs via weak interactions [vDW]
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5 Residues per turn in α-helices
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Reducing gent breaks Disulfide bridges in α-helice of keratin
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–Grow & Divide
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–Die [Apoptosis]
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–Requires cells to be touching
...
WBC integrins interact with EC ICAMs
Secretion: Molecules travel through environment and affect target cells
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e
...

-Steroid hormones [sex]: Oestrogen, Testosterone & Progesterone [produced in gonads]
->Oestrogen: diffuses across plasma membrane [hydrophobic], binds to nucleus receptor activating TFs
Hsp90 prevents the receptor from activating in Oestrogen absence
...
–Binds to RAR [cytoplasmic receptor] crossing into nucleus
Binds to RARE [RA response element], regulating gene expression
...

Autocrine: Cells respond to signalling produced by themselves [self-stimulated]
T lymphocytes respond to Antigens by secreting Growth Factors which increase their own proliferation
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Synaptic signalling- Neurons and synapses
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Arginine in blood converted to NO in EC ->NO diffuses into local SMCs
NO binds to Guanylyl Cyclase -> GTP converted to Cyclic GMP
Smooth muscle cells relax, and blood vessels dilate
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–Hydrophilic molecules bind to cell surface receptors
...
e
...

->Receptor types: Ligand-ion gated= Ligand [ion] binds to receptor
...
–Adjacent to that= Heterotrimeric G protein [active/inactive]
Membrane bound effector protein
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–Binds to inactive catalytic domains bridging 2 domains closing them together
...
–Cytoplasmic domain attached binds to the hetero-G protein
GPCR activated- GTP binding protein: α & γ bound to intracellular side of membrane, β sits on γ domain
...

In the absence of a signal, the α subunit has a GDP bound therefore the G protein is inactive
...

GTP replaces GDP in α-subunit activating G protein- causes conformational change in α-subunit & βγ complex
GTP-bound α-subunit binds to target membrane protein activating it -> causes signalling cascade in the cell
...
–RGS accelerates process
...

Enzyme converts ATP -> cAMP [2nd messenger]
cAMP activates PKA [protein Kinase A] by binding to its regulatory subunit
...

=->Deregulation: Glioblastoma Multiforme- Aggressive brain tumour, increase PKA level x10
...
–Gα activates Phospholipase Cβ [enzyme] -> cleaves PIP2 generating IP3 & DAG [2nd messenger]
...
–Mobilises Ca2+
...
–Protein phosphatase removes P from molecule
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–Serine
...

-Growth Factor= RTK ligands
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Nerve growth factor [NGF]: Neurotrophin, regulator of development and neuron survival
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Fibroblast proliferation- regenerates damage tissue
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Receptor Kinase principle: Ligand binds to ligand binding site of RTK -> Dimerization/Phosphorylation of Lip tyrosines activating them
ATP converted to ADP -> Additional phosphorylation [adding of phosphate] to Tyrosine residues
...
->Tyosine phosphorylation occurs- cytoplasm
->Insulin, dimeric receptor, binds to α-domains on receptor -> Receptor Tyrosine Kinase phosphorylated causing cellular response to insulin
=>Proteins with SH2 domains bind to Phosphorylated Tyrosine
...
–GAP
...
–Grb2
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–These Adapters bind to the SH2 domain of receptors [PDGF-β, EGF]
...
–GBR2 binds to Phosphorylared Tyrosine residues on EGFR via its SH2 domains
GBR2 also has an SH3 domain
...
–N terminus= Gab1, C terminus= Sos
...

Sos then binds to Ras [bound to GDP] on the the cell membrane
...

When GTP binds, Ras activates
...

Activated MEK 1/2 & ERK 1/2 activate Transcription factors [AP-1 family which are Fos and Jun]
-Direct: MAPK [ERK] dimers enter nucleus -> Phosphorylates Ternary Complex Factor [TCF]
-Indirect: MAPK phosphorylates p90Rsk kinase -> p90Rsk enter the nucleus -> Phosphorylates serum response factor [SRF]
=> TCF + 2SRF -> Trimer which binds to SRE [serum response element] on DNA molecule -> Fos gene transcription
GTP is hydrolysed leading to dissociation of GTP and Ras from Raf
-Genes which code for Ras= K-ras
...
N-ras
...
–ERK= MAP kinase
...
It inhibits itself in the absence of signal
...
–Cell signalling depends on SH2 domains
IGF + Growth factor receptor -> Recruits and activates PI3-K -> PI3-K phosphorylates PIP2 generating PIP3
PIP3 recruits PDK Kinase to membrane -> PDK Kinase phosphorylates Akt/PKB which promotes cell proliferation
...
–Sculpt the body during normal development
-Process: Early- Mild convolution
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–Margination
...

- Breakage of nuclear envelope
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–Blebbing
...

Late- Phagocytosis of apoptotic bodies
...
Caspases: Cysteine proteases that cleave after aspartic acid
Pro-caspases: Inactive precursors activated by cleavage
...
–Phosphatidylserines are exposed
...

-Response to injury/stress: Lack of Oxygen
...
–DNA damage
...
–Lack of extracellular survival signals
->Intrinsic pathway [Mitochondria] -> Cytochrome C -> Caspases -> Apoptosis
...

Apoptosis- Activation of Cysteine Proteases [Caspases], cleave after Aspartic acid residue
...

Apoptosome recruits Procaspase-9 via CARD [caspase recruitment domain] -> Caspase 9 is activated ->
Caspase-9 Cleaves & activates Procaspase-3 [executioner] -> Caspase 3 kills the cell
...
–Bcl2 binds to BH123 on mitochondria membrane and inhibits the aggregation
...
–FasL usually on T lymphocyte
...

A Death inducing signalling complex is formed [DISC] -> Initiator Caspases cleave DISC activating Executioner Caspases
Executioner Caspases activate Endonucleases -> DNA fragments -> Cytoskeleton caspases also break down the cell structure
...
–Normal P53 induces expression of Fas-R [apoptosis promoter]
Induces expression of IGFBP3[Insulin growth factor binding protein], outside the cell it binds and activates IGF which Inhibits proliferation
P53 drives Bax expression which activates intrinsic apoptosis pathway

LECTURE 25- THE CELL CYCLE
Purpose: Cell to grow and divide
...
–Cells are responsive to Mitogens and Growth factors & inhibitor
S [DNA synthesis]
...
–Preparation of cell division
M[Mitosis]
...
–Microtubules form mitotic spindles
...

–Metaphase: Chromosomes become fully attached to the spindles
...

–Telophase & Cytokinesis: Chromosomes decondense back
...

Promoting Proliferation: Cyclin dependant kinases [Cdk1, 2, 3, 6]- remain in the cell in an inactive state waiting to be activated by Cyclin
...
–Cyclin binding causes the T-loop to move [partial activation]
Partially active Cdk is then fully activated via cdk-activating kinase [CAK]
->Cyclins regulate CDK complexes
...
–E= 2 [late G1]
...
B=1 [G1 phase]
->Cyclins drive the cell cycle
...
–CDK4 and 6 act in the G1 phase/synthesis
=>DNA content marks the stage of the cell cycle
...

Cdks regulated by Phosphorylation: Wee1 kinase phosphorylates Active Cdk which inhibits the molecule
...
–Removes the extra Phosphate used to inhibit Cdk from Wee1
M-Cdk1 has a positive feedback on Activating Phosphatase Cdc25 inhibiting it
->Cyclin B: Enters G1 phase -> Binds to CDC2 in the S phase but complex is non-functional -> Cyclin B/CDC2 complex phosphorylation ->
Thr 161, Tyr 15, Thr 14 are inhibiting inactivation still -> CDC25 then enters removing the Inhibiting residues [only when at mitosis]
Cells cannot leave Mitosis step until Cyclin B has been removed and degraded
...
Of condensins
...
lamins
...
GM130
...

Cyclin B is targeted by Anaphase promoting complex [APC/c]
...

This process marks Cyclin B for degradation by a Proteosome
...
–Half way in G1 phase, point ‘R’, cycle is [internal influ
...

Bind to RTK producing TF factors like Fos and Jun ultimately producing D1 cyclin
->Mitogens control cell cycle: Phosphorylate P21 and P27 through PI3K- Akt/PKB pathway causing cytoplasmic localisation therefore inhibition
TGFβ [transforming growth factor] strongly activates P15, weakly induces P21
LECTURE 26- CELL CYCLE CONTINUED
The cell cycle control system: Checkpoints= Restriction ‘R’ point [Between G1 and S phase]
...
–Cyclin levels vary depending on the position of cell cycle
->MPF- maturation promoting factor, advances cell from G2 to M phase when the respective CDK molecule is bound to Cyclin
...

->G2 checkpoint, checks for DNA damage, or mis-match bases from replication
...

=>R checkpoint: pRb= regulator protein for Restriction point Gate
...

Activity is controlled by Phosphorylation -> Cyclin D binds to Cdk 4/6 activating the kinase
...

At the end of Mitosis; pRb is dephosphorylated via PP1, hence reactivating pRb causing suppression of cell proliferation
...

->pRb proteins inhibit E2F (1,2 & 3) transcription factor
...

pRb is inhbited via Hyperphosphorylation; E2Fs are free to move promoting passage into S phase
...
–Build-up of Cyclin E[Cdk2] phosphorylates pRb inhibiting it
...
->Further cell progression
...
–Defective RB gene; thickening of optic nerve & displacement of normal retina
...
–UV radiation
...
–Oncogene signalling
...

P53 will set to: Repair DNA
...
–Induce Apoptosis of cell
...
->These kinases Phosphorylate P53
-> Mdm2 [ubiquitin ligase] typically bound to P53, targets it for degradation during proliferation
...

-Loss of P53: Cancer- Ionising radiation -> DNA damage, tumour cell with no p53 -> No cell arrest -> Cell division with damaged chromosomes
...

=>M to A checkpoint: Cyclin A/B [Cdc2] helps APC activation [Anaphase promoting complex] -> APC causes Securin degradation ->
Separase is free -> Allows separation of sister chromatin by cleaving Cohesin which holds them together
...
-> Inhibited Cdc25 prevents Dephosphorylation of Cdc2
Cdc2 remains phosphorylated therefore Inactive -> No progression of G2-M phase
...
–E7 [inhibits pRb]
...


LECTURE 27- BIOINFORMATICS
Bioinformatics: The management and analysis of Biological information via the utilisation of computational techniques and technology
...
–Functional genomics: Understand function of genes, determine their roles
...
–Proteomics: Study of structure, function & protein interaction
-Metabolomics: Study of small molecules [sugars, nucleotides, amino acids, lipids]
...
–Partial sequence of a clone [300-500bp]
...

Databases: DNA= Genbank
...
–DDBJ
...
-MIPS
...
–TrEMBL
NCBI: National centre of biotechnology information- information source of all genetics and genomics disciplines
...

–Tries to deduce genes biological function to sequence data
...
–ORF; coding sequence from start to end
...
–BLAST
...
–GENSCAN
BLAST- Basic local alignment search tool
...

Paralogy= Homogolous genes within a single species which diverges by gene duplication
...
List of hits which have statiscal similarity to query sequence
...

Proteomics: Proteins with related function
...
->35%= common 3D structure
...
Information of gene traits and disorders
...
–Completed in 2003
...
–DNA databases: GenBank
...
–DDBJ
...
–PIR
...

Morgan: [1920s] Fruit fly genetics study; isolates mutations which affect visible traits
Giemsa staining can give rise to bands but no observable gene mutation
...
–Immunohistochemistry
...
–Tumour/blood samples [stained]
...
–Purification
...
–Visualisation via staining
...

-> Tissue homogenisation -> Cell lysis -> Protein removal -> Organic extraction of Nucleic acids -> Purification
...

–RNA and DNA have different properties and chemical groups requiring them to be separated and purified by different means
...

-Gel electrophoresis: DNA separation [-]
...
–Polyacrylamide= Complex, higher resolution
...
–Can detect Sickle cell anaemia; point mutation which alters the restriction site
DNA region with 2 restriction sites: Point mutation deletes 1 restriction site
...

-Identifying gene mutations: Southern blot [1975]: After gel electrophoresis -> Blot onto Nylon – makes a permanent record of DNA separation
Absorbed DNA on Nylon is hybridized with allelic probe
Stringency or ‘conditions’ must be optimum for max hybridisation
LECTURE 29- PCR: DNA ANALYSIS
PCR: 1985
...
–Template always 3’-5’
...

-Components: x10 Buffer [mg2+]
...
–dNTPs [50-200uM]
...
–DNA polymerase
...
–Reverse transcriptase activity [Tth]
...
–Complementary to 3’ end
...

Primers bind to the 3’ flanking regions of both DNA templates
...
–Porphyrin/ haem groups act as inhibitors for PCR
...

->Optimisation: Timing
...
–Cycle number
...
–Ampliwax [separates reagents]
PCR enhancers- Helix destabilisers
...

=>Magnesium concentration: Low mg2+= low yield
...

Mg2+ serves as a cofactor for Taq Polymerase, assists in strand stability
...

-PCR sensitivity/contamination: Uracil-N-Glycosylate kit [UNG], Uracil becomes incorportates
...
–RFLP
...
–Real Time PCR
...
–Mutation detection: ASO-PCR, SSCP
...

-RT-PCR: Extract RNA [has PolyA tail] -> Anneal a PolyT primer -> Extend with RT -> cDNA made
–High quality RNA needed, useful for Expression analysis
...

->RT-PCR: uses Less RNA
...
–SSCP: Single stranded conformational polymorphism [uncharacterised mutation]
Characterised mutations- ASO-PCR [Allele specific oligonucleotide]
...
–Optimisation [temp, pH, glycerol, ions]
...
–Hot SSCP; radiolabelled
...
–Specific ASO primer used [30 nucleotides]
...

->ASO-Multiplex: Uses 2 sets of primers in the PCR [2 ASO-primers]
->Real-time PCR: Quantifies PCR products
...
For analysis]
...
–The more dsDNA accumulates, the more dye binding occurs
...
–Non-specific DNA binding
...
-5’ nuclease assay, probe cleaved during PCR
...
–Reporter is released and detected in fluorescence
...
–Enzymatic [common & automated]
Primer directed synthesis of DNA
...
Chain terminators included
...

Extension phase uses Radioactive labels or a Dye Terminator
Chain termination= 4 steps -> A stop, C stop, G top, T stop
...

-4 Termination analogues form, with either A,C,G or T at the 3’ end
...
–Read from 5’ [bottom] to 3’ [top]
...
–Mutations, polymorphisms, Regulatory elements, and ORFs are identified
...
–Co-termination can occur; Polymerase stops at secondary structures
...

Missing bases [compression] due to dITP analogues
...

->Automated DNA sequencing: Fluorescent primer labelling [must be good quality and uniform]
...
–Capillary systems -> Long reads and high throughput
Fluorescent chain termination products will migrate down a single lane in the gel
Maxam & Gilbert ‘Chemical sequencing’: Harsh chemicals required
...
[RNA for some viruses]
...

–Reverse genetics: Genotype to phenotype [1980s]
...

-Positional cloning allowed to identify disease causing mutations [Cystic fibrosis, huntingtons, neurofibromatosis]
...
6kb] insert in a phage clone- used as a probe to detect RFLP
...

-End fragments of cloned inserts used to screen Genomic DNA library
...

-Leads to a contig of overlapping clones
...

->Contig: From contiguous [sharing a common border, i
...
end fragments] = A series of overlapping DNA sequences used to create a physical map
which reconstructs the original DNA sequence of a chromosome/region of chromosome
...
–Microsatellites [SSLP- single sequence length polymorphism, VNTR- Variable number tandem repeat]
...
-> Transfer DNA frag to Nylon mem
...
–Restriction endonuclease site present or not
...

->RFLP linkage mapping: RFLP segregate in genetic crosses as co-dominant genetic markers
...
of recombinants/total] x 100
...

Microsatellite markers: ‘SSRs’ simple sequence repeats or STRs [short tandem repeats]= repeating sequences of 2-5bp of DNA
...
–Typically Co-dominant
...

Dinucleotide repeats, mostly CA-GT
-Genotyping: Microsatellite alleles- genotyped with PCR amplification
...

Fluorescent detection used to identify microsatellites

Genome sequencing projects: Helped to radically advance Reverse Genetics
...
elegans
...

-1992: Human
...
–PCR approaches; Screening
->Human ESTs: Expressed sequence Tags- Approach via THCs [Tentative human consensus sequences- Contigs of overlapping EST]
LECTURE 33- COMPLETE GENOME SEQUENCE
D
...
Sequence: 120mb euchromatin
...
6K genes
...

-Dispersed DNA repeats: Segments of DNA which occur multiple times at random positions within the genome
Typically Transposable elements [large segments encoding a protein responsible for the moving the segments position]
Creates problems of True overlaps and Repeating overlaps
->True overlaps: Common segments between two fragments of a certain length -> The shared sequence involves fragments which come from
overlapping sections of the genome- the fragments belong together
...

-Unitigs/repeat boundaries: Unitigs are a uniquely assembleable subset of overlapping fragments
Assembly of fragments which have no competing choices in terms of internal overlaps
-> Either correctly assembled portion of contig or an overcompressed formation of many copies of repeats
...

=>Mate pairs: Eliminates dispersed repeat problem
...

Two short sequences of a clone, the ends of the sequences which both originate from the same clone are mate-pairs
...

Contigs can be linked together into scaffolds via connections between mate-pairs
Distance of contig can be determined by mate-pair positioning
->WGS technique: No physical map used
...

Scaffold is
Obtaining a random sequence from the genome
...
–Use RE to cleave DNA into many small segments
Take the small segments -> clone them into BAC vectors -> Cleave again into smaller fragments
...
–Requires high end automated algorithmic program [labour saving, program does the sequencing]
=>Disadvantages= Large requirement on ability to align overlapped fragments and construct the chromosome
...
9billion bp generated by WGS
->Only 1
...
-24% introns
...
9% identical DNA
...
1% makes us different
...

->SNP genotyping: Short oligonucleotide probes only anneal to target complementary DNA if it is a perfect match
...

LECTURE 34- HUNTINGTONS
Gene map for disease based on Positional cloning
...
–Linkage associated detected after many Trial and error probing
-RFLP marker location genotyped via probe G8
...

–Polymorphism detected by cutting DNA with HindIII generating different DNA fragment sizes [kbp]
...
5cM]
-DNA markers that are found to be tightly linked to mutant allele [<1cM] are used as molecular probes to screen genomic libraries for contigs
Chromosome walking: Maps large chromosome
...

Cut chromosome which restriction enzyme
...

Sequence gene A
...
–Use PCR to bind primer to DNA sequence at gene A
...
–Obtain subclone fragment of Gene a
...
–Apply primer to sequence and amplify again
...

Produces overlapping clones generated by partial digestion
...

Will identify the gene with the mutation
...
–SMBA
...
–Trinucleotide repeat of CAG in Huntington people= 36-100 copies
CAG repeat encodes Glutamine residues
...
–Aggregation eventually found in intranuclear inclusion bodies
...

=>Huntingtin: [Htt]- contains Glutamine/proline rich region at N terminus and 10 HEAT repeats clustered in 3 domains
...
–Apoptosis
...
–Cell signalling
...




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