Lecture 08 - Parasites of the genome... Viruses, plasmids,...

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Unformatted text preview: Parasites of the genome... Viruses, plasmids, & plasmids, genetic engineers Viruses are very degenerate parasites, with just a few genes. They simply inject their genomes into host cells, which replicate the virus. There is DNA coding for a protein coat, enzymes that insert the virus into and extricate it from the host DNA, and enzymes that direct its assembly into new viral particles. When many viral particles are made, the host cell explodes (lyses). (lyses). Viruses may interrupt important genes, and if they do not excise cleanly, can leave indels behind. Either of these can cause cancers (e.g., HPV & cervical cancer). Modified viruses can insert genes permanently, a technique used in some kinds of "gene therapy". therapy" Plasmids are even more degenerate. They are simply loops of "naked DNA", rather like a DNA" mitochondrial genome. They are common in bacteria (also found in plants). They are also relatively easy to manipulate, and favored in genetic engineering. Plasmids can pop in & out of bacterial genomes, or behave like small chromochromosomes. somes. 1 The dangerous thing about plasmids is that, a) some contain antibiotic resistance genes, and, b) they can be exchanged by bacteria, even across different species. "Restriction endonucleases" endonucleases" that defend bacteria against viruses are used by engineers for inserting genes into plasmids. plasmids. They cut at special "palindromic" palindromic" restriction sites, (e.g., GAATTC for "EcoRI"). EcoRI" Screening for uptake of modified plasmids: plasmids: Bacteria containing a modified plasmid: plasmid: Can be raised as a "library" library" (essentially a way of storing an organism of interest's genome) interest' Can be cultivated in tanks, and allowed to produce proteins Prior to DNA sequencing, you need to make some extra copies of the gene. So, there is one technique of which you should have heard at some point in a genetics course: PCR (the polymerase chain reaction). It is marginally more exciting than the whole-organism cloning procedure. whole- It is quite simple: make a cocktail that includes DNA polymerase, nucleotides, polymerase, the gene you want to copy, and very short pieces of DNA called "primers". primers" Heat up the mix until the DNA strands peel apart, then allow it to cool. As it does, primers bind to DNA first, stimulating replication starting from that point. Repeat 30 or so times, it makes a billion copies of the gene. 2 1. Heat (denature) 2. Cool (anneal) 3. Keep warm (replicate) 4. Each repetition preferentially copies DNA between primers. The inventor & the story of PCR are rather more entertaining than the actual process. 1) Kary Mullis, surfer/inventor dude from La Mullis, Jolla, conceived of PCR driving his Honda Jolla, Civic down Highway 128 from San Fran to Mendocino one foggy night. He won a 1993 Nobel, and is now on the talk circuit. 2) The denaturing step means you can't use can' just any old DNA polymerase... polymerase... To sequence DNA, add bases with dyes attached Screening for successful uptake of that preventinvolves application of antibiotics plasmids further extension plasmids carry antibiotic resistance (many of the strand. genes). Screening for successful insertion of gene into plasmid involves feeding bacteria a particular sugar that leaves a blue waste product (when gene is inserted in plasmid, plasmid, blue waste product is not produced). Thermus aquaticus (Taq) came from Yellowstone! Taq) Chimaera, Chimaera, hideous, firefirebreathing offspring of Echidna & Typhon, sister Typhon, of Cerberus & Hydra, and mother of the Sphinx, was slain by Bellerophon. Bellerophon. 3 The trick is to insert the marker gene in the right place... ... so that transcription and translation of the gene under study also results in the production of gfp, or luciferin, etc. gfp, luciferin, In 2002, we saw the first genetically engineered primate, ANDi (backwards acronym for integrated DNA). (He's (He' got a gfp gene.) The viral techniques used in animals are a bit more involved than modifications of plasmids used in plants & bacteria, but do essentially the same thing. Applications include: "pharming", e.g., pharming" producing human insulin in cow's milk; cow' gene therapy, replacing the function of the defective alleles with which people were born; modifying innocuous viruses to carry proteins from deadly ones. The Ti (tumor-inducing) plasmid (from (tumorAgrobacterium tumefasciens) is commonly tumefasciens) introduced into plants carrying the bt (Bacillus thuringensis) toxin gene, or extra thuringensis) copies of EPSP synthetase. synthetase. Genetic engineering of plants and bacteria is now commonplace, and unfortunately, wholly unregulated. Insecticidal & other genes are added to crops without testing for health effects. 4 ...
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