Lecture 12 10 1 12 Mitosis 1 In G2 DNA 2x then M where it divides and goes back to X Interphase chromosomes are relaxed G1 G2 Microtubules that form spindles move the chromosomes Pull sister chromatids apart and partition them 2 Metaphase chromosomes have condensed and formed sister chromatids Chromosome being pulled by microtubules to other sides while lined in the middle Visualization of Chromosomes Interested in chromosome patterns 1 Begin with growing cells to observe cell in mitosis Must be rapidly growing Only small fraction of the cells will actually be in mitosis Must maximize cells in mitosis to observe chromosomes Beginning of mitosis chromosomes condense Fibers microtubules begin to pull chromosomes 2 Colchicine plant extract 12 20 hours Will disrupt microtubules to fall apart into their subunits G1 and G2 will continue onto replicating and into mitosis If in Mitosis cells will freeze in mitosis because spindle fibers cannot move Population of cells in Mitosis will increase in number increasing the chance of seeing chromosomes in mitosis 3 Must break apart suspension of cells Mash cells over slide Wash with methanol acetic acid Removes lipids and makes DNA accessible 4 Use trypsin solution continuing protease digests some o the proteins holding chromosomes together 5 Giemsa stain same enzyme phosphorylating lamina will also cause the chromosome to condense Activates enzyme that causes centromeres to split chromsomes to split by phosphorylation MPF gather growing cells incubated with colchicine cells in mitosis prophase accumulate use methanol acetic to remove lipids Trypsin to digest proteins centromere Giemsa stain Special Karyotyping SKY Look for unique sequences t o chromosome 1 Synthesize segments stretches DNA complementary to the chromosome using a DNA sysnthesizer Oligonucleotides 35 base pairs Now have accumulated 24 pairs of these sequences from 24 chromsomes including Y On chromosome 1 link dye that fluoresces yellow Dye ataches covalently Chromosome 5 all oligonuclueotides will fluoresce yellow orange Chromosome 8 will fluoresce light yellow Must denature DNA to help separate and relax double helix Translocation piece of chromosome that migrates to another chromosome Chronic Myeloid Leukemia progressive First shows itself as increase in number of leukocytes granula Over period of 5 years chronic phase leukocytes continue to increase without any intervention Organs begin to become compromised kidney failure or respiratory infection Decrease in density of bone cells and increased in number How to recognize disease early on Karyotype analysis Bone marrow biopsy Pattern Nowell Hungerford 1960 called Philadelphia chromosome Shortened chromosome 22 reciprocal translocation between 9 and 22 Mosaic Gene BCR ABL codes for protein tthat is a mosaic Chrom amino end to carbonyl end BCR ABL protiens Proteins unable to be regulated always on Some drugs selectivey inhibit CDK cyclin Gluvec BCR from chromosome 22 ABL from chrom 9 Lecture 13 10 3 12 Proteins can cross in a selective manner specified by proteins embedded in membrane Not always energy requiring process can be used to regulate physiological functions Movements across membrane Diffusion At equilibrium concentrations inside and outside the vesicle cell match Simple diffusion no carrier channel Nothing intervening between substance and membrane Carrier mediated diffusion some sort of protein embedded in membrane speeds up process of diffusion What determines rate of entry and exit Rate of collision to membrane V out to in proportional to concentration outside of cell V flux proportional to number of particles area micrometers squared seconds S0 outside S O inside Net flux V V out to in V in to out K S0 S 0 k diffusion coefficient Find rate of exit and entry to be linear Measuring Rate of Entry Using whole cells 1 Take sample of RBCs that have been isolated in solution containing glucose 2 RBCs require glucose for cell processes 3 Allow to incubate for as little as possible 1 minute 4 Collect cells and measure glucose concentration inside RBC compared to initial concentration some cells already have glucose in cells How do you measure glucose in cells Use radioactive form of glucose Or generate Ghosts Place RBCs in water RBC s will swell up and enzymes and internal structures will leak out Place back in isotonic solution to regain normal size RBC now is empty except with transport membranes Or generate artificial membranes Simple vs Facilitated Diffusion Simple linear line Facilitated rate maximizes and will level off Becomes saturated One of the clear indications of facilitated diffusion is that you will get saturation Channel glycerol uptake in E coli Has aqueous channel to allow movement of specific molecules Glycine E coli bacteria plasma membrane cell wall Glycerol moves with mechanism involving a carrier Found to be protein embedded in plasma membrane glycerol channel Found to be aquaporin water channel Periplasm Channel consists of alpha helix spanning membrane loop with alpha helix 6 helices that span membrane and 2 loops 4 subunits to make 4 channels Loops inside channel determine specificity of channel space between cytoplasm and plasma membrane 6 alpha helices give wall of cylinder 2 loops one from cytoplasm end one from extracellular end inside channel presence of internal loops provide specificity Serves as transport of water in kidneys and urine excretion Found in fat cells Degaration of lipids require movement of water Mutations in aquaporins resulted in fat mice Same channel allows glycine urea straight chain sugars Glycerol CH2 OH CH OH CH2 OH Glycine H2N CH2 COOH Urea H2N C O NH2 Experiment by creating mutation block entry of urea glycerol glycine and sugars into cell through transport membrane Since glycerol and glycine use same channel rate of entry of glycerol will decrease Without glycine present glycerol has normal facilitated diffusion curve With glycine present if rate of entry changes we will know they use the same carrier What will the presence of competitive inhibitor do to a curve Modeling rate of entry Subcellular vesicles Lecture 14 10 5 12 Aquaporin homotetramers Selectivity doors 10 9 molecules can move through per second Facilitated Diffusion Direction of equilibrium Channels Saturable Mutation could block transport Determine whether facilitated or simple through kinetic movement Movement of glucose into mammalian cells ligand specific carrier Erythrocyte suspended in blood
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