Biol 4610 1st EditionExam # 2 Study Guide Lectures: 6 - 13Lecture 6Methylation of DNA – another epigenetic modification Similar to histones, when we methylate DNA we decrease transcription This happens on cytosine Opposite side of methyl group forms the hydrogen bond w Guanine Important in development DNA methylation represses transcription -By “masking” a TF binding site Transcription factors recognize promoters- specific DNA sequences of ~10bp in length Ezh2- histone methylase DNMT- DNA methyl transferaseChapter 8 – 8.4. Post-transcriptional Gene ControlTo control translation after transcription, can use ~20-26bp short RNAs:1) Those that repress translation of specific mRNAs (called microRNAs or miRNAs)1) Small ribosomes2) Those that cause mRNA degradation (termed short interfering RNAs or siRNAs). 2) MicroRNAs or miRNAs:1) These are present as genes (>1000)2) Can be transcribed like any other gene (have promoters, enhancers etc.)3) Primary microRNA (70-80bp in length)4) Forms a pseudo-complementary (imperfect) binding5) The folding back on itself allows it to be bound in the nucleus by a protein called DGCR8 (double stranded RNA binding protein)6) Drosha- ribonuclease that cuts part of microRNA2) RNA Interference (siRNA) – need a perfect match in the 21-23bp long siRNA. – Come from viruses or are manufactured in lab– Rather than binding to 3’ translating region, they bind to the coding area of mRNA. Need perfect match of base pairs to get this to work.– Argonaute gets activated and cuts mRNA. (only works if perfectly complementary). Cuts into pieces of ~10bp in length In nucleus we have genes that code for miRNADrosha does some cutting and gets exported out into nucleusCan also have siRNA, made artificially or from a virus.Both mRNA and siRNA must be cleaved by a dicer. Once cleaved, they get loaded into RISC complex.Is RNA is mismatched, miRNA – 3’ UTRIs perfect complementarity – siRNA- mRNa gets chopped upBottom line à decreases protein productionLecture 7Chapter 99.1. Growing cells in CultureNutrient requirements – glucose, amino acids, vitamins, salts,1. EUKARYOTIC CELLS NEED GROWTH FACTORS• Proteins that are used to signal a cell to divide• Often come from serum2. Sterility- inside a cabinet, keep out bacteria so divide slower. Also must be at appropriate temperatureSolid supports – most eukaryotic wont grow in Suspension.Petri dish but put a coating like anExtracellular matrix.Polarity or sidednessPrimary cultures – arise from tissue• Finite lifespan- can only replicate so many times Immortal cell lines – grow forever in culture1. In rodents- this can happen spontaneouslyMost die off but a small proportion of them will continue to grow. So we can get them to stay in culture and use them for the next experiment.2. Chemical treatment (mutant DNA)3. Tumor cells, use cancer biopsy and get them to grow forever Separate cells using a flow cytometer or fluorescence activated cell sorter (FACS) –If want a pure culture, we need a way of separating out the cells we don’t want.With the antibody, the Y shaped region recognizes a particular protein that is on a virus.This protein is on the cell of interest (skin cell), but not on another cell type (immune cell). So we can use fluorescently labeled antibodies to recognize the skin cell and stick to them and go thru a straw. Straw only allows 1 cell to pass at a time. Light bulb detects what’s fluorescently labeled by antibody and separates it from the rest. 9.2. Light microscopyNormal bright-field light microscope: • Has a light bulb and uses different lenses to condense light and magnify objectResolution:• See objects down to 0.2 microns• If have 2 objects closer together than 0.2 microns, won’t be able to distinguish them andwill look like 1 object.Phase-contrast microscope:Uses difference in thickness of cellsRefractive index: • light moves more slowly in thicker areas• Ex. If have giant nucleus, light will bend in areas that are thicker• “light moving out of phase”• Areas look darker if out of phase or in thicker areasGood for: • Visualizing single layer cells in culture• Thin tissue slicesNot good for:• Thick tissues, everything will be bentDifferential interference contrast (DIC) microscopes:• Based upon the thickness of cells• Refractive indexFixing and staining cells• Cells have few macromolecules that absorb light well• Other forms of light microscope are not good for thick samples (better for single layers)• Use different stains so it’s easier to look at• “Fix” cells with Formaldehyde- cross links DNA – proteins so macromolecules aren’t degraded• Thick samples: embed in paraffin and then slice into thin sectionsHematoxylin and eosin (2 diff dyes)  Hema. Is basic- (proteins) used for nuclei and stains blue or purple Eosin binds to negatively charge/acidic. Labels the cytoplasm with pink/red colorLecture 8Chapter 10 – Biomembrane structure- Define the cell- Separate inside from outside- Define organelles- Transporting- Signaling- Adhesion to other cells- Structure/interaction with cytoskeleton 10.1. Phospholipid Bilayers: Lipid Composition and Structural Organization3 types of lipids in most membranes:• Phosphoglycerides- stabilized by hydrophobic (vanderhaals) interactions and H bonds• Exoplasmic face: outside of cell• Cytoplasmic face: inside of cell• Phosphate group attached to Glycerol 3C and 2 fatty acyl chainsDiffering head groups on the phosphoglyceride Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinostiol (PI)PC: most common head group (neutral at physiological pH)2) Sphingolipids – derived from sphingosine• 1 acyl chain is attached to glycerol by amide linkage• Makes the membrane thicker (because it’s longer relative to phosphoglyceride)3) Cholesterol – a 4-ringed hydrocarbon. • Also considered amphipathic • Used to add fluidity to membrane. If have all long chains, they pack together and are in arigid state. Cholesterol adds fluidity to this.Lecture 1011.3 ATP powered pumps and intracellular Ionic EnvironmentActive transporters All require the ATP hydrolysis to functionGroup into 4 different classes of pumps:1. P-class pumps – move ions up the concentration gradient; move a variety of ions2. V- class – pump hydrogen ions; used for acidification; lysosomes3. F- class – pump hydrogen ions; ATP synthase molecule; use H