Lecture 8.docx

Lecture 8 Carbon Fixation – CO2added to RuBP to produce two 3PGA molecules Reduction – NADPH and ATP used to convert 3PGA to G3P, a higher energy molecule to build sugars Calvin Cycle  A characteristic of the Calvin Cycle is that it makes ATP. FALSE (uses) At the Nucleus  Telomerase – DNA Polymerase in eukaryotes that extends the end of the lagging strand  See sheet  To start replication you need an RNA primer to start replication by DNA Poly  DNA Poly uses free 3` OH on RNA to begin DNA polymerization  RNA has to be removed and it leaves a gap o Telomerase (protein + RNA) will replicate the lagging strand end Video (on Moodle) – DNA Replication  Each new DNA is semiconservative  DNA Helicase breaks the helicies apart and the point where it is is called the replication fork  ssDNA (less stable) are protected by single stranded binding proteins (SSB’s) o called “Stabalizing proteins”  RNA Primase makes the RNA Primer required for DNA Poly to start o From 5` end only one primer needed (leading strand) o From 3` end primers required sequentially because it works backwards nd  2 DNA Poly the removes the RNA primers puts in the new DNA  DNA Ligase synthesizes the gaps between fragments of DNA because DNA Polymerase can’t o Note these gaps are called Okazaki Fragments  RNAse removes the RNA primers E. Coli and Molecular Bio  Selecting for plasmids  Look at Biotech documents BIOTECHNOLOGY Plasmids  Only are necessary for when they are selected for  Extrachromosomal DNA  Often carry antibiotic resistant DNA Genetic Engineering  Ori – origin of DNA replication (different than the genomic ori) o One genomic ori in bacterial chromosomes o Many in eukaryotes  Marker allows us to select for bacteria with plasmid o E.g. amp resistant genome “bla” (β-lactamase which degrades amp)  Promoter is the area that generally attracts and begins the replication  MCS – on plasmids used for genetic engineering o Not in nature, is synthetic sequence o Stands for Multiple Cloning Sequence o Where enzymes (restriction enzymes) can be used to cut plasmid  insert foreign DNA into the cut site  Transformation – put plasmid DNA into cells o Frequency is about 1:10,000 Organisms used in Biotech  E.g. food is biotech Use of Bacteria  The Introns stay within the DNA and thus the correct proteins that won’t work  Hard to get into because of Lipopolysaccharide (herd for DNA to pass though)  High protein [] leads to cell death o So the extra proteins made on the plasmid, if expressed could cause cell to die and the actual experiment will fail Eukaryotic Systems  CHO – Chinese hamster ovaries  BHK 1 – Baby hamster kidney  They can be modified properly and thus the Introns that have to be spliced are spliced  BUT animal calls are hard to culture in the lab o Expensive and time consuming Yeast  Eukaryotic! (for beer and bread)  Has haploid (one set/copy of each chromosome) o For humans, the haploid # is 23  Also has diploid (two sets/copies of each chromosome) o For humans, 46  Can undergo meiosis (Diploid  Haploid) or mitosis (diploid  diploid)  Can do post transcriptional (splicing, adding 5` methyl G-cap) and post-translational modifications  But: o Expression is low – can’t make a lot of engineered protein o Pattern of modification can be different Plants  Plant cell lines are very similar in post-transcriptional/translational modifications with humans  Very easy and cheap to culture  “killer genes in pollen” – often genes are inserted into the plant behind pollen specific promoters o Thus expressed only in pollen which will kill the cell so that it can’t reproduce  Problems: o Patents – contamination o Can have allergies o Long growing Bacterial Genomes  Virulence – ability to infect and cause damage o Some virulence genes are separate, some are together in plasmid Antibiotics  Some kill things like DNA Gyrase (a topoisomerase that makes or breaks supercoi