Part #2 - R.Roy - Lecture 17 October 22nd Molecular Genetics II(pages 182189 198208 Cloning A group of organisms produced from one stock or ancestor The

Part #2 - R.Roy - Lecture 17 October 22nd Molecular...

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Unformatted text preview: Lecture 17 – October 22nd Molecular Genetics II (pages: 182 ­189; 198 ­208) Cloning: A group of organisms produced from one stock or ancestor The Cloning of Dolly  ­ Was cloned from a single nucleus that was put into another enucleated cell (nucleus was destroyed) Gene Cloning: Cloning through the plasmid ­cloning vector Map the mutated gene and isolate the DNA corresponding to the mutant gene and compare it to wild type (normal form) of DNA and conclude what those DNA protein ­encoding region might be doing in a normal organism. • Clone the mapped mutated gene fragment DNA and put it into plasmids • Use restriction enzymes to cut the DNA sequence and plasmid at specific sites in a specific manner forming sticky ends (overhangs) • Polylinker region were engineered to have multiple restriction sites • Digestion of a vector with two different enzymes facilitates directional cloning so that the differentially digested DNA fragments that you are interested in can only be inserted in one orientation Studying genes of interest: DNA libraries Permanent collections of genes can be obtained and maintained in DNA libraries  ­ Different libraries are used depending on the information you want to study  ­ Genomic libraries contain copies of the DNA present in the genomic/chromosomal contact (introns, exons, repetitive sequence, intergenic regions) – ALL of the DNA  ­ cDNA libraries represent mRNA (tissue specificity, abundance) mRNA can be enriched from a starting population of total RNA • Isolate all RNAs • mRNA is the least abundant form of RNA • It can be separated from RNA through an affinity column that’s based on specific interactions – gel linked to a poly ­T ­oligonucleotide  The poly (A) tail of mRNAs enables their purification via oligo ­T ­ nucleotides linked to a solid support. The poly (A) tail will stick to the oligo ­T ­nucleotide and the other RNAs will go through. Isolating the mRNA population.  Using the oligo ­T paired to poly (A) tails as a primer, reverse transcriptase (isolated from retroviruses) can synthesize a DNA strand complementary to an mRNA, the complementary or cDNA  Extends poly ­T 3’ end  End with RNA ­DNA hybrids mRNA can be converted to cDNA using retroviral Reverse Transcriptase 1. mRNA is converted to cDNA by priming the poly A tail with a single ­stranded poly T oligonucleotide – binds to 3’ end 2. RT uses this primer to initiate single ­strand DNA synthesis that is fully complementary to the mRNA template – RNA ­DNA hybrid 3. Alkali or RNaseH then removes RNA and a poly dG adapter is annealed to the 3’ end. 4. A poly dC primer is used to initiate synthesis of the second DNA strand by recognizing and binding to the poly dG adapter 5. E.coli DNA polymerase I progresses through any remaining hybrid regions and extends the second strand using first strand as template Studying genes of interest: Genomic and cDNA libraries  ­ cDNA libraries correspond to the population of mRNA molecules present at any given time, or in a given tissue (or both), which can be generated and plated out  ­ Are a permanent, reusable record of the mRNA expressed in a given tissue/stage  ­ Every single mRNA (cDNA) should be represented at least once among the phase plaques.  ­ Can amplified by plasmid vectors or bacteriophages  ­ You want to have as many different cDNA molecules as possible  ­ Complexity allows for the low abundance transcripts to be represented Studying genes of interest: RT ­PCR (reverse transcriptase based PCR)  ­ PCR greatly facilitates the exponential amplification of target DNA molecules from a minimal amount of starting template  ­ Since all mRNAs present in a given cell will have a poly A tail then simply poly T primers can be used to prime a strand of cDNA using RT ­PCR  ­ The resulting cDNA molecules in the reaction solution after several cycles should be a reasonable prediction of the various starting levels of each species  ­ PCR is not a hundred percent faithful – cDNA libraries are better Bacterial expression vectors: over expression of recombinant protein  ­ Specialized vectors that function in E.coli can be used to overexpress recombinant proteins of biomedical interest ie. Insulin The lac promoter provides an effective means of inducing gene expression. • Take the lac promoter and put it in a specific vector with the gene we are interested in downstream of that promoter – kick out lacZ gene • Introduce vector into bacteria and induce the expression of wanted protein in large quantities Sometimes this is tight enough – proteins expressed are quite toxic We can use this same technology in a bacteriophage: • Contains a T7 polymerase gene downstream of lac operon • Gene is kept off • Can drive expression of the gene of interest or, in many systems it can drive T7 RNA polymerase gene to be transcribed, which in turn drives expression of the recombinant gene under the control of a T7 promoter  IPTG induce transcription • T7 RNA polymerase binds to T7 RNA polymerase binding sites and transcribes extremely fast  ­ Dormant bacteria phages – lyzogens  cell often die due after Specialized vectors permit efficient expression in higher eukaryotic cells Transfect a vector into eukaryotic cells  Electroporation – opens pores in the cells  Lipid treatment of cells • Expression vectors must have an origin of replication and a promoter a) Transient transfection: only some cells express the transgene  Vectors get into the cells but don’t integrate and are eventually eliminated  For a couple of days you can examine the expression of the vector b) Stable transfection: all the cells express the transgene (+ antibiotic resistance)  Vector has a strong drug resistance gene  Select for those cells that integrated the vectors into the chromosomes • Lecture 18 – October 24th Expression of fusion proteins facilitates functional analysis  ­ Jelly Fish (Aequorea Victoria) produces a fluorescent protein that has been named Green Fluorescent Protein (GPF)  ­ A fusion protein can be generated between any given promoter or protein and GFP so that the “GFP ­tagged” protein will be fluorescent  ­ GFP ­tagged histones and tubulin decorate the chromosomes and the cytoskeleton during mitosis in a living cell in real time  ­ This protein is used to see when a cell is producing a certain protein by replacing it with GFP and observing when the cell turns green Optogenetics approaches take advantage of the properties of channelrhodopsin  ­  ­  ­  ­  ­  ­  ­ Channelrhodopsin integrates into the membrane and makes proteins sensitive to a specific wavelength of light When you shine that light on it – there’s a conformational change in the protein and it acts as a channel, which allows ions to flow through it It has the ability to depolarize cell Took the channelrhodopsin gene and cloned it into various vectors that were introduced into organisms Can be used to drive neurons and make them sensitive to light stimulation Potentially could be used in the medical field Mouse experiment – neurons that affected feeding behaviors had channelrhodopsin and made the mouse eat repeatedly Molecular biological techniques advance our analytical capabilities Qualitative Analysis:  ­ The nature of the molecule(s) in question  ­ Size?  ­ Nucleotide composition?  ­ Conformation/configuration?  ­ Structure? Quantitative Analysis:  ­ Molecular approaches can be used to determine, quite precisely, the levels of specific gene products • I.e. tumor markers Molecular probes can be used to find a needle in a haystack A complex mixture of macromolecules Binding to a solid phase support Probe specific target recognition Remove non ­ specific Target Detection       Mixture of macromolecules include proteins, mRNA, rRNA – crushed up cells Take mixture and put in on agarose gel to separate molecules via MW Take everything out of gel and transfer to a solid phase matrix – nitrocellulose or nylon – things cannot move Add probe, which allows you to specifically identify target of interest – DNA probes or RNA probes, etc… Wash like crazy to get rid of non specific things and leave things that stick Detect probe through necessary means – fluorescent probes, radioactive Single stranded oligonucleotides can be labeled using polynucleotide kinase to act as a probe – are ordered for experiments  ­ Known sequence that corresponds to gene or gene product of interest  ­ If you only know the protein sequence: Synthesize an oligonucleotide that has the complementary sequence to the specific region of the gene (cDNA) of interest ­this may be a “degenerate pool –wobble base pairs on 3rd codon  ­ Polynucleotide Kinase (PNK) will phosphorylate nucleotides by transferring the γ phosphate of ATP to the free hydroxyl at the 5’ end of the single stranded oligonucleotide – if γ phosphate is marked with some sort of isotope, after this reaction the oligonucleotide becomes very radioactive  ­ If you get rid of all unused radiolabeled ATP and purify only the radioactive oligonucleotide – you can identify the sequence that are complementary to it PCR can be used to make radiolabelled DNA probes  ­ Take a DNA template with a region of a gene that you are interested in  ­ Need an upstream and downstream primer  ­ By incorporation deoxyribonucleotides that carry a radiolabel on the α ­ phosphate into PCR amplified DNA, specific DNA molecules can be very efficiently labeled  ­ Unincorporated radioactive substrates are subsequently removed and the radiolabelled DNA can then be used as a probe  ­ It must be rendered single stranded prior to use after purification Analysis of nucleic acids by transferring to solid ­state supports (nylon, nitrocellulose  DNA; nitrocellulose  RNA)  ­ Both DNA and RNA can be separated according to size using an agarose gel.  ­ DNA is cut with a restriction enzyme and then run through an agarose gel • A diagnostic signature that reflects the DNA sequence  ­ mRNAs of different sizes will correspond to the various genes that encode them  ­ Both of these analytical techniques require that the molecules be transferred to a solid state support (nylon/nitrocellulose) and be denatured (single stranded) for subsequent analysis  ­ Molecules are then transferred to the filter paper and covalently bound Southern Blot Once covalently bound the levels and positions are permanently recorded  ­ The nucleic acids are the bound covalently to the support  ­ This permanently records the levels (abundance) and the position (size) of the molecules following separation on the gel  ­ The support or “blot” can then be “hybridized” with probes to any sequence that may be of interest  ­ Washes remove non ­specific signal and only complementary sequences will be detectable on the blot following autoradiography Nucleic acid hybridization techniques enable DNA and RNA detection  ­ Molecular identification of RLPs can detect disease through MW of alleles  ­ RNA detection uses northern blotting This is important for doing analysis of genomes/genotypes Key Points:  ­ Molecular biological analyses allow us to make important quantitative and qualitative conclusions about any gene of interest  ­ Complementary molecular probes are important tools to identify (blots) or isolate (libraries) nucleic acid sequences of interest  ­ Many of the molecular biological techniques used routinely rely on the ability of nucleic acids to form DNA ­DNA, RNA ­DNA and RNA ­RNA hybrids  ­ Genomics and PCR have facilitated many of these techniques Protein Structure, Function and Separation Strategy Lecture 19 – October 26th (pages 77 ­80; 88 ­99) Crystallography and X ­ray diffraction have been useful to identify protein structure because it’s hard to predict them from amino acid sequence  ­ High concentrations of purified protein can organize to form crystal lattices by playing with conditions to favor their formation  ­ These crystals can be bombarded with high energy beams (i.e. X ­rays) which are scattered according to the atomic arrangement of the atoms within the crystal  ­ The scatter pattern is detected by radioactive detectors and the data is analyzed by complex programs to provide a prediction of electron density Electron density maps provide a skeleton upon which one can build a model  ­ By recording the electron density maps structural biologists can begin to build models by filling in the observed density with amino acids structures that are known to correspond to such shapes  ­ Amino acids are linked into ball and stick models to complete the chain and then secondary and tertiary structures along with individual interactions (hydrogen bonding, van der Waals interactions…) between residues can be highlighted Proteins can be catalytic enzymes  ­ Enzymes reduce the energy required to carry out a given reaction  ­ Enzymes must recognize a substrate in its substrate binding site  ­ Also require an independent catalytic site, which can be close to substrate binding site or far from it since folding will bring it closer  ­ Often enzymes enhance substrate interactions such that the reactions are favored  ­ Critical residues in the active site may be involved in ternary complex formation and drive reactions forward Quantification of catalytic efficiency can reflect specific properties of a protein  ­ Catalytic efficiency can depend on the substrate type – not all will be used at the same rate – depend on how the substrate fit in substrate binding site  ­ The more substrate that you add to a limited amount of enzyme will give rise to a certain amount of product until you arrive at a plateau – saturation  ­ The half max Km – doesn’t ever change for a given enzyme or substrate – is the substrate concentration at which that enzyme works a 50% of its max Proteins can bind other molecules  ­ Proteins may interact specifically with other molecules  some small molecules, lipids, other proteins or small peptides  ­ Some common ligands include: • Growth factor  small polypeptides that will interact with receptors • Steroid hormone  small lipids that bind to complex receptors • Cytokines  small molecules that bind to receptors  ­ Ligand binding can change protein conformation considerably: Allosteric switches  ­ Strength of a protein/affinity of protein interaction can be expressed as: • Kd – Dissociation Constant = [A]*[B]/[A ­B] Calcium, Calmodulin and Conformation (Allosteric Change)  ­ Many proteins require Ca2+ for optimal function. Often requires a Ca2+ binding protein called Calmodulin  ­ Calmodulin radically changes its conformation when bound to Ca2+  ­ In the Ca2+ bound state Calmodulin can recognize specific regions of proteins to which it binds  thereby altering protein function in a Ca2+ dependent manner Proteins can act as switches/triggers  ­ Active proteins often exist in an “on” and “off” state and fluctuate between these states in response to intra ­ or extracellular cues  ­ When GTPase is bound to GTP – the trigger is ON  ­ When the protein is bound to GDP – the trigger is OFF  ­ GTP binding proteins can possess a GTP hydrolyzing activity (GTPase) that converts GTP  GDP  ­ This activity is enhanced by GTPase activating proteins (GAPs). The GDP is efficiently displaced by GTP due to the Guanine exchange proteins (GEFs)  ­ Phosphorylation by protein kinases can also act as a trigger by catalyzing changes in protein conformation – add charge by adding phosphate  ­ These changes are counteracted by protein phosphatases that remove these modifications Centrifugation – Purifying proteins SDS ­Polyacrylamide Gel Electrophoresis  SDS ­PAGE Western Blots 2 ­Dimensional (2d) Gel Electrophoresis – separation by charge  ­ Stop migrating once they have arrived at their neutral point First separate using pH gradient according to their charge, then separate them according to their size. Gel filtration or size exclusion chromatography separates according to size  ­ Matrix contains pores or indents of a specific size (Agarose, Sephadex)  ­ Protein fraction is loaded onto the column and individual proteins or complexes will flow through the channels between the beads and/or through the pores  ­ Small proteins make it through the pores and hang out in the matrix while big proteins are confined to space between the beads  ­ Very large proteins simply flow through the column while smaller proteins take the long way – bigger proteins leave earlier  ­ Longer column lengths provide greater separation efficiency Ion exchange chromatography: separation based on electrochemical properties – based on electrochemical properties of proteins  ­ Matrix can be negatively or positively charged Affinity chromatography  ­ The most efficient method of purifying cloned gene products  ­ In a DNA, construct an in frame affinity tag can be added to engineer a translational fusion variant of any protein of interest  ­ This may faci...
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