BSCI330 Exam 2 notes Lecture 10 1 Karyotype Analysis cells in mitosis become visible to count chromosomes abnormality breakage helps identify malignancy condensed form How to Detect Chromosome Stages of Eukaryotic Cell after mitosis before split into two daughter cells G2 S M 2x x S G1 G2 cells 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 phase Phases 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 division Visualization of Chromosomes growing 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 frozen mitosis prophase fix cells in methanol drop suspension of cells on to slide smash on cover slip and add MAA which eliminates some lipids and makes DNA more available acetic acid trypsin solution 10 15 min protease digests proteins that held DNA together stain 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 chromosome MPF Mitosis promoting factor cyclin CDK1 staining pattern unique for chromosomes Spectral Karyotyping double stranded DNA denature DNA so relaxes condensation synthesize segments complementary to those stretches linker dye so all oligonucleotides will fluoresce identifying chromosome uniquely incubate cells with mixture of oligonucleotides and dip chromosomes in complementary pairs will attach sensitive to translocations Chromosome 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 CML Mosaic 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 proliferate SOLUTION Gluvec blocks ABL inhibits kinase cell proliferation and bone marrow transplant Lecture 10 3 Movements Across Membrane 1 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 process How 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 molecules must bump each other into the membrane rate of bump movement proportional to concentration area in 2 x sec How 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 inside Saturation Kinetics Vmax reach saturation by inc conc of glucose inc carriers channels can reach saturation only certain amount of carriers facilitated can reach saturation Models 1 Whole cell messy because may have some glucose already measure glucose C14 and watch and count radioactivity 2 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 shape 3 Artificial Membrane make artificial membrane in test tube Channel glycerol uptake in E coli aqueous channel for specific mol cell wall and plasma membrane are diffusive barrier amino acids like glycine or alcohol like glycerol could be tested carrier mediated facilitation mutation could block glycerol sugars urea and glycine from entering aquaporins 6 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 competition Lecture 10 5 Facilitated Diffusion non concentrative flux in direction of equilibrium channels saturable mutation could block transport glycerol channel facilitator protein Ligand specific carrier glucose translocator protein Physiological regulation of facilitated diffusion glucose translocation in adipocytes water movement through kidney glucose transport in erythrocytes glucose binds into protein and then released 5 mM glucose concentration moves into cell embedded in membrane and recognizes ring to exit structure Glycolysis Glucose Pyruvate glucose hydrolyzed by hexokinase phosphorylated on C6 glucose 6 phosphate phosphorylation of glucose retains unidirectional how Can t go back out transporter specific for glucose single direction in erythrocytes retained by
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