The composition of chromatin and comparison of genome sizes (very broadly) for viruses, prokaryotes, and eukaryotes (level discussed in lecture).chromatin = DNA + proteinsViruses Prokaryotes (E. Coli) Human5.1 kb (base pairs) 4,000 kb (base pairs) 2,900,000 kb (base pairs).0017 mm (long) 1.36 mm (long) 990 mm (long)cell only 2*10^-6 by .8 µm nucleus is 10*10^-6 m ? HU: bending of chromosome, basic proteins that stabilize structuresupercoiling due to 2 topoisomerasessimple- cut apart strands and unwind then religate forced to twist on itself, torsion created1 histones: .7 nonhistone(for 1 g DNA about 1 g histone + nearly 1 g nonhistone)The classes of histones and the general traits/features of these proteins.- highly conserved in all animals- basic due to amino acid side chains (positive at neutral pH), attracted to negative DNA- lysine and arginine- 8 molecules (2 of H2A/B, 2H3, 2H4) make up core- H1 is separate and helps tighten binding- nucleosome is 110 Å and wraps 147 bp DNAThe folding or levels of condensation of a eukaryotic chromosome starting with DNA and through the architecture of a metaphase chromosome, plus the experimental basis behind the information (at the level of details discussed in lecture and related reading).- DNA double helix- beads on a string chromatin- solenoid or zig-zag - section of chromosome in extended form- condensed section of chromosome- entire mitotic chromosome (most highly condensed)result: each DNA is packaged into a mitotic chromosome and condensed 10,000 foldhow to examine the nucleosome:1. isolate unfolded chromatin by digestion with nucleases that cut between nucleosomes2. exposed DNA between nucleosomes (linker regions) is degraded3. dissociate DNA from core via high ionic solutions4. measure length of the DNA associated with that nucleosome5. run the sections of DNA on a gel, DNA from 2 nucleosome will travel 2x as much as DNA from 1 nucleosome etc. low ionic concentration: beads on a string, beads of 100 Åhigher ionic concentration: 300 Å fiber (solenoid or zig-zag)high ionic concentration: DNA dissociates completely from histone core, looped domains + scaffold which is nonhistone proteinHow would a sample of cells obtained from an individual be examined for potential chromosomal abnormalities using karyotype analysis [details of the assays and the reasons behind each of step]when cell is about to enter mitosis, chromosomes condense, spindle fibers enrich for cells in metaphase:1. extract fluid from growing fetus, want to look at their chromosomes, the cells in mitosis*few cells that are proliferating are in mitosis (mitosis is only 20 min. of 20-24 hour long process)2. increase # of cells in mitosis by colchicine3. fix cells in methanol-acetic acid4. trypsin for 10-15 min. 5. stain with giemsa, 6. smash cells in mitosis so chromosomes will spread at random by dropping at a specific height7. take picture and cut them apart by size and banding pattern organize them*cholchicine: (incubate for 20 hr) chromosomes are stuck in metaphase, binds to monomers of microtubules and keeps them from polymerizing so spindle fibers don’t formspectral karyotyping/chromosome painting: procedure combines elements of above process up until staining chromosome, does not use giemsa- 46 chromosomes chose one at random- sequences on each chromosome is known- complementary nucleotide sequences (oligonucleotides) covalently attach a fluorescent dye that can appear in computer- incubate isolated chromosome with mixture of oligos instead of gimesa- oligios specific to parts of the chromosome after incubation, different fluorescent markers can bind*used to identify structural chromosome aberrations in cancer cells and other disease conditions when Giemsa banding or other techniques are not accurate enoughGive examples of different forms of facilitated diffusion that allow the movements of molecules througha) an aqueous channelwater soluble molecules (water) not ionsglycerol ureaglycineb) via a carrier.ligand specific carrier: glucose translocator proteinglycolysis maintains unidirectional entry of glucose in the cell! makes it appear as though there is less glucose in the cell so direction is to move inside, unidirectional flow through hexokinases Discuss the selectivity of each type of translocator and contrast the structural differences of each class of translocators.6 alpha helices span the membrane and have 2 dangling loopsgreen = alpha helicesyellow is core of channel where substances can passGLUCOSE TRANSPORTERS1. first level of regulation- type of translocator, families of genes that code for proteins (GLUT)GLUT 1 & 3 GLUT 2 GLUT 4brain, heart, smooth muscles liver, pancreas striated muscles, fatty cellskM=1mM kM=15-20 km kM=5mMblood glucose concentration is 5mMfunction maximally even in small concentrations, high affinity for glucoseonly when glucose if very high will the carriers be functioning optimallydifferent GLUT ensure there is adequate distribution of glucose to all the specific tissues2. second level of regulation- 12 alpha helices, provide wall of a cylinder (larger than aquaporin)slight amino acid differences affect affinities & determine specificity as glucose enters channel - H bonds between H and Oxygen on glucose and some of the side chain molecules of amino acids which provide specificityHow would you determine whether the transport of the amino acid glycine into red blood cells occurs by simple diffusion or by facilitated diffusion? If glycine were to be imported by facilitated diffusion, how could you determine whether or not another amino acid (such as methionine) moves into the cells by the same carrier?1. add to suspension a concentration of amino acid whose diffusion you want to measure, do this at different concentrations of amino acids2. run the experiment at different concentrations3. measure over time intervals4. finally pellet the cells and count how many amino acids there are in them(label the amino acids with radioactive tags) so they can be counted ex. amino acid methionine labeled as 35S-methioninefinally graph the results and the trend will suggest the type of diffusionfacilitated diffusion is faster because there is an actual hole for it to go through as opposed to working its way through the lipid bilayerRBC ghosts:1. put RBC into water so they will expand and contents will leak out2. place them in isotonic solution and resume original shape3. vacant cell is created you know exactly what it contains+ no internal
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