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Recombinant DNA Andy Howard Introductory Biochemistry 14 October 2010 Biochem: Recombinant DNA 10/14/2010 Recombinant DNA Much of our current understanding of molecular biology, and of the ways we can use it in medicine, agriculture, and basic biology, is derived from the kinds of genetic manipulations that we describe as recombinant DNA Biochem: Recombinant DNA 10/14/2010 Page 2 of 60 What we’ll discuss High levels of DNA structure (concluded) Synthesis of DNA in the laboratory Cloning Plasmids & inserts Vector techniques Libraries & probes Biochem: Recombinant DNA High-throughput Expression Fusion Proteins Protein-protein interactions 10/14/2010 Page 3 of 60 Nucleosome structure Core octamer is two molecules each of H2A, H2B, H3, H4 Typically wraps around ~200bp of DNA DNA between nucleosomes is ~54 bp long H1 binds to linker and to core particle; but in beads-on-a-string structure, it’s often absent Biochem: Recombinant DNA 10/14/2010 Page 4 of 60 How much does this coil up? 200 bp extended would be about 50nm The width of the core-particle disk is 5nm So this is a tenfold reduction Nucleosomal organization corresponds to negative supercoiling … so DNA ends up supercoiled when we take away the histones Biochem: Recombinant DNA 10/14/2010 Page 5 of 60 Courtesy answers.com Next level of organization H1 interacts with DNA along linker region Individual histones spiral along to form 30 nm fiber See fig.19.25 Courtesy Johns Hopkins Univ Biochem: Recombinant DNA 10/14/2010 Page 6 of 60 Even higher… The 30nm fibers are attached to an RNA-protein scaffold that holds the 30nm fibers in large loops Typical chromosome has ~200 loops Loops are attached to scaffold at their base Ends can rotate so it can be supercoiled Biochem: Recombinant DNA 10/14/2010 Page 7 of 60 What about prokaryotes? No actual histones Histone-like proteins (HLPs) involved Bacterial DNA attached to scaffold in large loops (~100kb) This makes a nucleoid Biochem: Recombinant DNA 10/14/2010 Page 8 of 60 How many loops in bacteria? Typical bacterial genome (E.coli) has 3000 open reading frames ~ 3000 genes. Assume 500 amino acids per protein = 1500 bases per gene (ignores transcriptional elements) Then genome is 1500 bp/gene * 3000 genes = 4.5*106 base-pairs That’s (4.5*106 bp)/(1*105 bp/loop) = 45 loops Biochem: Recombinant DNA 10/14/2010 Page 9 of 60 iClicker quiz, question 1 1. How does acetylation of histones affect their charge state? (a) It makes them more positively charged (b) It makes them less positively charged (c) It does not change their charge state (d) It depends on whether these are bacterial histones or eukaryotic histones Biochem: Recombinant DNA 10/14/2010 Page 10 of 60 iClicker quiz, question 2 2. Suppose a mutation in the gene coding for histone H1 makes it fold up incorrectly. How will this mutation influence DNA organization? (a) It will prevent formation of nucleosomes (b) It will interfere with the beads-on-a-string organization between nucleosomes (c) It will interfere with higher-level organization involving assembly of solenoids into loops (d) All of the above (e) None of the above Biochem: Recombinant DNA 10/14/2010 Page 11 of 60 Synthesizing nucleic acids Laboratory synthesis of nucleic acids requires complex strategies Functional groups on the monomeric units are reactive and must be blocked Correct phosphodiester linkages must be made Recovery at each step must high! Biochem: Recombinant DNA 10/14/2010 Page 12 of 60 Solid Phase Oligonucleotide Synthesis Dimethoxytrityl group blocks the 5'-OH of the first nucleoside while it is linked to a solid support by the 3'-OH Step 1: Detritylation by trichloroacetic acid exposes the 5'-OH Step 2: In coupling reaction, second base is added as a nucleoside phosphoramidate Biochem: Recombinant DNA 10/14/2010 Page 13 of 60 Synthesis I Figure 11.29 Solid phase oligonucleotide synthesis. The four-step cycle starts with the first base in nucleoside form (N1) attached by its 3'-OH group to an insoluble, inert resin or matrix, typically either controlled pore glass (CPG) or silica beads. Its 5'-OH is blocked with a dimethoxytrityl (DMTr) group (a). Biochem: Recombinant DNA 10/14/2010 Page 14 of 60 Blocking groups If the base has reactive -NH2 functions, as in A, G, or C, then N-benzoyl or Nisobutyryl derivatives are used to prevent their reaction (b). In step 1, the DMTr protecting group is removed by trichloroacetic acid treatment. Step 2 is the coupling step: the second base (N-2) is added in the form of a nucleoside phosphoramidite derivative whose 5'-OH bears a DMTr blocking group so it cannot polymerize with itself (c). Biochem: Recombinant DNA 10/14/2010 Page 15 of 60 Solid Phase Synthesis Step 3: capping with acetic anhydride blocks unreacted 5’-OHs of N-1 from further reaction Step 4: Phosphite linkage between N-1 and N-2 is reactive and is oxidized by aqueous iodine to form the desired, and more stable, phosphate group Biochem: Recombinant DNA 10/14/2010 Page 16 of 60 Activation of the phosphoramidate Biochem: Recombinant DNA 10/14/2010 Page 17 of 60 Cloning Cloning is the process whereby DNA is copied in a controlled way to produce desired genetic results Biochem: Recombinant DNA 10/14/2010 Page 18 of 60 Plasmids Small (typically < 10 kbp), usually circular segments of DNA that get replicated along with the organism’s chromosome(s) Bacterial plasmids have a defined origin of replication and segments defining specific genes Some are natural; others are man-made Biochem: Recombinant DNA 10/14/2010 Page 19 of 60 How they’re used Typical man-made plasmid includes a gene that codes for an enzyme that renders the bacterium resistant to a specific antibiotic, along with whatever other genetic materials the experimenter or clinician wishes to incorporate Thus the cells that have replicated the plasmid will be antibiotic-resistant; surviving colonies will be guaranteed (?) to contain the desired plasmid in all its glory Biochem: Recombinant DNA 10/14/2010 Page 20 of 60 A typical plasmid Biochem: Recombinant DNA 10/14/2010 Page 21 of 60 Building useful plasmids Take starting plasmid and cleave it with a restriction enzyme at a specific site Add foreign DNA that has been tailored to fit into that plasmid Biochem: Recombinant DNA 10/14/2010 Page 22 of 60 Inserts Typically a place within the plasmid will be set up so that small stretches (< 10 kbp) of desired DNA can be ligated in With sticky ends: high specificity, but you do get self-annealing of the plasmid and of the insert, so those have to be eliminated With blunt ends: require more artisanry: T4 phage ligase can rejoin ends without stickiness; but it’s chaotic Biochem: Recombinant DNA 10/14/2010 Page 23 of 60 Directional cloning Guarantees that the desired DNA goes in in exactly one orientation Biochem: Recombinant DNA 10/14/2010 Page 24 of 60 Use of bacteriophage lambda Can handle somewhat larger inserts (10-16 kbp) Middle third of its 48.5-kbp chromosome isn’t needed for infection Biochem: Recombinant DNA 10/14/2010 Page 25 of 60 Cosmids 14-bp sequence cos (cohesive end site): 5’-TACGGGGCGGCGACCTCGCG-3’ 3’-ATGCCCCGCCGCTGGAGCGC-5’ … one of these at each end Must be 36 kbp < separation < 51 kbp apart In practice we can use these for inserts up to 40 kbp in size Biochem: Recombinant DNA 10/14/2010 Page 26 of 60 Cosmids in action (fig. 12.9) Biochem: Recombinant DNA 10/14/2010 Page 27 of 60 Shuttle vectors These are plasmids that can operate in two different organisms Usually one prokaryote and one eukaryote (e.g. Escherichia coli and Saccharomyces cerevisiae) Separate origins for each host This allows us to clone the vector in a bacterial host and then express it in a eukaryotic setting Biochem: Recombinant DNA 10/14/2010 Page 28 of 60 Typical shuttle vector Biochem: Recombinant DNA 10/14/2010 Page 29 of 60 Artificial chromosomes Huge chunks (2 megaBp!) can be propagated in yeast with artificial chromosomes (YACs) These can be manipulated in the yeast setting or transferred to transgenic mice in a living animal YACs need origin, a centromere, and telomeres Biochem: Recombinant DNA 10/14/2010 Page 30 of 60 Use of YACs in mice QuickTime™ and a decompressor are needed to see this picture. Biochem: Recombinant DNA 10/14/2010 Diagram courtesy Expert Reviews in Molecular Medicine, 2003 Page 31 of 60 DNA libraries Set of cloned fragments that make up an organism’s DNA We can isolate genes from these Most common approach to creating these is shotgun cloning, in which we digest the total DNA and then clone fragments into vectors Goal is that >= 1 clone will contain at least part of the gene of interest (might have been clipped by the restriction enzyme!) Biochem: Recombinant DNA 10/14/2010 Page 32 of 60 Probabilities Probability P that some number of clones, N, contains a particular fragment representing a fraction f of the genome: P = 1 - (1 - f)N Therefore 1-P = (1-f)N Thus ln(1-P) = ln{(1-f)N} = Nln(1-f) Therefore N = ln(1-P) / ln(1-f) Biochem: Recombinant DNA 10/14/2010 Page 33 of 60 What that means The value f is pretty small, so the denominator is only slightly negative; whereas we want the numerator to be very negative, since that corresponds to a high value of P. 10 kbp fragments in E.coli means f = 10/4640 = 0.0022, so for P = 0.99, we need N=1.4*106 We’d do better with larger f values! Biochem: Recombinant DNA 10/14/2010 Page 34 of 60 Finding relevant fragments by colony hybridization Plate out a library of fragments and grow colonies or plaques Soak those onto a flexible absorbent disc Disc is treated with high-pH to dissociate bound DNA duplexes; placed in a sealed bag with a radiolabeled probe If they hybridize, radioactivity will stick to disc The hits can be recovered from the master plate Biochem: Recombinant DNA 10/14/2010 Page 35 of 60 Colony hybridization illustrated Biochem: Recombinant DNA 10/14/2010 Page 36 of 60 Making the probes Sometimes we have at least part of the gene sequence and can fish for it Other times we know the amino acid sequence and can work backward, but with degeneracy (64 codons, 20 aa’s) Typically use at least 17mers to guarantee that the don’t get random association Probes derived from a different species are heterologous With big eukaryotic genes we may have to look for pieces of the gene, not the whole thing Biochem: Recombinant DNA 10/14/2010 Page 37 of 60 cDNA libraries Sometimes the easiest thing to obtain are mRNA templates associated with a particular function Reverse transcriptase can make a complementary (cDNA) molecule from such an mRNA template A library of cDNAs can be assembled from a collection of mRNA templates Biochem: Recombinant DNA 10/14/2010 Page 38 of 60 Why is that useful? The mRNAs will be unique to the cell type from which they’re derived Often they’re also unique to the functional role that tissue is playing at the time Therefore finding that collection of DNA tells us about cellular activity Biochem: Recombinant DNA 10/14/2010 Page 39 of 60 Expressed sequence tags An EST is a short (~200 base) sequence derived from a cDNA Represents part of a gene that is being expressed Labeled ESTs can be mounted on a gene chip and used to identify cells that are expressing a particular class of mRNAs Biochem: Recombinant DNA 10/14/2010 Page 40 of 60 Southern blots I: fractionation Tool for identifying a particular DNA fragment out of a vast population thereof Exploits sequence specificity for identification Developed by E.M.Southern in 1975 Begins with electrophoretic fractionation of fragments (mobility 1/mass) Polyacrylamide gels ok 25-2000 bp; agarose better for larger fragments Biochem: Recombinant DNA 10/14/2010 Page 41 of 60 Southern blots 2: blotting Gel soaked in base to denature duplexes pH readjusted to neutral Sheet of absorbent material placed atop the gel Salt solution is drawn across the gel, perp to the electrophoretic direction, in various ways to carry the DNA onto the sheet Sheet is dried in an oven to tightly attach the DNA to it Incubate sheet with protein or detergent to saturate remaining DNA binding sites on sheet so we don’t get nonspecific binding Biochem: Recombinant DNA 10/14/2010 Page 42 of 60 Southern blots 3: hybridization Labeled probe and sheet placed in sealed bag If probe attaches, label will appear at that point on the sheet via annealing or hybridization Label detected by autoradiography Biochem: Recombinant DNA 10/14/2010 Page 43 of 60 Southern blots illustrated Biochem: Recombinant DNA 10/14/2010 Page 44 of 60 Variations on this idea RNA can be used as the probe: that’s called a Northern blot Proteins can be substituted by using an antibody as a probe and a collection of protein fragments as the analytes; that’s called a Western blot Ha ha There is no Eastern blot, as far as I know. Biochem: Recombinant DNA 10/14/2010 Page 45 of 60 High-throughput techniques Eagerness to provide rapid, easy-to-use applications of these approaches has led to considerable research on ways to make these techniques work fast and automatically This high-throughput approach enables many experiments per unit time or per dollar Biochem: Recombinant DNA 10/14/2010 Page 46 of 60 DNA microarrays Thousands of oligonucleotides immobilized on a substrate Synthesis by solid-phase phosphoramidite chemistry Typically 25-base oligos Can be used in cDNA projects to look at expression patterns Biochem: Recombinant DNA 10/14/2010 Page 47 of 60 An example Biochem: Recombinant DNA 10/14/2010 Page 48 of 60 Using expression vectors We often want to do something with cloned inserts in expression vectors, viz. make RNA or even protein from it RNA: stick an efficient promoter next to the cloning site; vector DNA transcribed in vitro using SP6 RNA polymerase This can be used as a way of making radiolabeled RNA Biochem: Recombinant DNA 10/14/2010 Page 49 of 60 Protein expression Making (eukaryotic) proteins in bacteria via cDNA means we don’t have to worry about introns Expression vector must have signals for transcription and translation Sequence must start with AUG and include a ribosome binding site Strong promoters can coax the bug into expressing 30% of E.coli’s protein output to be the one protein we want! Biochem: Recombinant DNA 10/14/2010 Page 50 of 60 QuickTime™ and a decompressor are needed to see this picture. Example: ptac This is a fusion of lac promoter (lactose metabolism) with trp promoter (tryptophan biosynthesis) Promoter doesn’t get turned on until an inducer (isopropyl--thiogalactoside, IPTG) is introduced Biochem: Recombinant DNA 10/14/2010 Page 51 of 60 iClicker quiz, question 3 Probe systems employing RNA are called (a) Southern blots (b) Northern blots (c) Western blots (d) Eastern blots (e) None of the above Biochem: Recombinant DNA 10/14/2010 Page 52 of 60 iClicker quiz, question 4 4. The inducer used with the ptac promoter system is (a) glucose (b) glucose-6-phosphate (c) IPTG (d) ionizing radiation (e) none of the above. Biochem: Recombinant DNA 10/14/2010 Page 53 of 60 Eukaryotic expression Sometimes we need the glycosylations and other PTMs that eukaryotic expression enables This is considerably more complex Common approach is to use vectors derived from viruses and having the vector infect cells derived from the virus’s host Example: baculovirus, infecting lepidopteran cells; gene cloned just beyond promoter for polyhedrin, which makes the viral capsid protein Biochem: Recombinant DNA 10/14/2010 Page 54 of 60 Screening libraries with antibodies Often we have antibodies that react with a protein of interest If we set up a DNA library and introduce it into host bacteria as in colony hybridization, we can put nylon membranes on the plates to get replicas of the colonies Replicas are incubated to make protein Cells are treated to release the protein so it binds to the nylon membrane If the antibody sticks to the nylon, we have a hit! Biochem: Recombinant DNA 10/14/2010 Page 55 of 60 Fusion proteins Sometimes it helps to co-express our protein of interest with something that helps expression, secretion, or behavior We thereby make chimeric proteins, carrying both functionalities We have to be careful to keep the genes in phase with one another! Often the linker includes a sequence that is readily cleaved by a commercial protease Biochem: Recombinant DNA 10/14/2010 Page 56 of 60 Fusion systems (table 12.2+) Product Origin Mass, Secre Affinity Ligand kDa ted? -galactosidase E.coli 116 No APTG Protein A Staph. 31 Yes IgG Chloramphenicol acetyltransferase E.coli 24 Yes Chloramphenicol Streptavidin Strep. 13 Yes Biotin Glut-S-transferase E.coli 26 No Glutathione Maltose Bind.Prot. E.coli 40 Yes Starch Hemoglobin 16/32 No None Vitreoscilla Biochem: Recombinant DNA 10/14/2010 Page 57 of 60 Improving purification via expression If we attach (usually at the N-terminal end) a his-tag (several his, several cys) to our protein, it becomes easier to purify: The his tag forms a loop that will bind strongly to a divalent cation like Ni2+ Thus we can pour our expressed protein through a Ni2+ affinity column and it will stick, while other proteins pass through We elute it off by pouring through imidazole, which completes for the Ni2+ and lets our protein fall off Biochem: Recombinant DNA 10/14/2010 Page 58 of 60 Protein-protein interactions One of the key changes in biochemistry over the last two decades is augmentation of the traditional reductionist approach with a more emergent approach, where interactions among components take precedence over the properties of individual components Protein-protein interaction studies are the key example of this less determinedly reductionist approach Biochem: Recombinant DNA 10/14/2010 Page 59 of 60 Two-hybrid screens Use one protein as bait; screen many candidate proteins to see which one produces a productive interaction with that one Thousands of partnering relationships have been discovered this way Some of the results are clearly biologically relevant; others less so Biochem: Recombinant DNA 10/14/2010 Page 60 of 60