BSCI330 Exam 2 notesLecture 10/1Karyotype Analysis- cells in mitosis become visible to count chromosomes #, abnormality, breakage- helps identify malignancy, condensed formHow to Detect Chromosome #Stages of Eukaryotic Cell (after mitosis before split into two daughter cells) G2 2xS M x ------------- S G2 G1cells rapidly proliferate- G1 = begin proliferations proliferation, begin replication of DNA- S = doubles DNA content- G2 = splits chromosome → 2 daughter cells - liver cells will proliferate if damaged- present in G1 (waiting for stimulus to start process)- karyotype viewed in M phase- stem cell research taken from infants because cells ready to proliferate in G1 phasePhases of Mitosis- microtubules = fibers that can pull two sister chromatids apart- polymers of microtubules fall apart and become fibers to split sister chromatids (metaphase)- form spindles and serve as tracts for vesicles- during mitosis fibers in cells breakdown to form after divisionVisualization of Chromosomesgrowing population of cells = (only small fraction in mitosis, procedure wants to maximize chromosomes in mitosis)colchicine 12-20 hrs = (added to solution after cells have been rapidly growing, disrupts microtubules no matter what stage the cell is in, inhibits proliferation and cell's don't die)accumulate cells in = all phases will continue except cells in M phase will be frozenmitosis (prophase)fix cells in methanol- = drop suspension of cells on to slide, smash on cover slip and add MAA whichacetic acid eliminates some lipids and makes DNA more availabletrypsin solution 10-15 min = protease, digests proteins that held DNA togetherstain with Giemsa = stain(colchicine used in cancer treament because stops cells from proliferation)When looking under microscope- many cells with many nuclei on slide- chromosomes spread on surface- cut chromosomes- look at lengths, staining, #, to detect which chromosomeMPF – Mitosis promoting factor- cyclin β – CDK1- staining pattern unique for chromosomesSpectral Karyotyping- double stranded DNA- denature DNA so relaxes condensation- synthesize segments complementary to those stretches- linker dye so all oligonucleotides will fluoresceidentifying chromosome uniquely - incubate cells with mixture of oligonucleotides and dip chromosomes in- complementary pairs will attach - sensitive to translocationsChromosome 18 – translocation = piece of chromosome broke off and lygated to other chromosome Chronic Myeloid Leukemia (Nowell and Hungerford)- density of cells much smaller- lethal and progressive disorder- increase in number of leukocytes (not alarming unless persists for a few months)- increase for 5 years without intervention then WBC are so high and organs are destroyed (secondary disoder)- ex. kidney failure because too many cells and proteins are released taking up too much space for other cells - early detection through Bone Marrow biopsy◦ cells taken → caused to proliferate → view under microscope → if chromosome 22 is shortened and reciprocal translocation with 9 and 22 = indicates CMLMosaic gene #22: BCR ● ABL: #9- when BCR and ABL come together gene is always on- amino (NH3) | BCR|ABL| (COO-) carboxylic- kinase indicates cells to proliferateSOLUTION- Gluvec blocks ABL (inhibits kinase/cell proliferation)- and bone marrow transplantLecture 10/3Movements Across Membrane1) Diffusion- moves toward equilibrium- not energy costing- redistribution of molecules across membrane- simple◦ equilibrium reached with time◦ concentration of molecule inside and out are equal- carrier-mediated◦ protein embedded in membrane that speeds up processHow to tell:Passive Diffusion:- flux = # of particles moving across certain area over time - constant related to shape of molecule/temp- rate is linear - in order for substances to move, moleculesmust bump each other into the membrane- rate of bump movement proportional to concentration- area in μ^2 x secHow to Measure the Rate of Entry- RBC in solution with glucose- want to generate linear model based on concentration difference across cell-surface- saturation kinetics- leveling off with facilitated diffusion, linear with simple diffusion Experiment - suspension of whole cells → at point zero add concentration of glucose in solution and add radioactive substrate → incubate cells for few sec.s because you want enough glucose to enter without having too much in gradient → collect and measure glucose concentration → set up experiment again with different concentration of glucose outside vs. insideSaturation Kinetics = Vmax- reach saturation by inc. conc ofglucose = inc. carriers- channels can reach saturation- only certain amount of carriers- facilitated can reach saturationModels1.Whole cell- messy because may have some glucose already- measure glucose C14 and watch and count radioactivity2. Subcellular vesicles- take RBC put in water and they begin to swell and contents leak out- empty vesicle an then be put in solution to regain it's shape3. Artificial Membrane- make artificial membrane in test tubeChannel: glycerol uptake in E. coli- aqueous channel for specific mol- cell wall and plasma membrane arediffusive barrier- amino acids like glycine or alcohollike glycerol could be tested- carrier-mediated facilitation- mutation could block glycerol, sugars, urea and glycine from entering- aquaporins◦ 6 α-helices make cylinder, 2 selectivity loops (in cytoplasm and extracellular)- mutations of gene coding for glycerol channel in mice makes mice obese because degradation of lipids involves movement of glycerol out of fat cells and if glycerol can't move mice become fat- glycerol, d-manitol, d-sorbitol, d-ribitol all can pass through channel because geometry of channel◦ TEST: mutating gene for glycerol channel and all molecules will be effected because none can pass through now◦ TEST: competition, add glycine and rate of glycerol will be slower b/c of competitionLecture 10/5Facilitated Diffusion - non-concentrative- flux in direction of equilibrium- channels- saturable- mutation could block transport (glycerol channel/facilitator protein)Ligand-specific carrier - glucose translocator proteinPhysiological regulation of facilitated diffusion - glucose translocation in adipocytes- water movement through kidney- glucose transport in erythrocytes◦ glucose binds into protein and then released to exit◦ 5 mM glucose concentration moves into cell ◦ embedded in membrane and
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