The bacterium Escherichia coli (or E. coli) is a single celled organism that lives in the gut of healthy humans and animals. Its body shape can be modeled as a 2-?m-long cylinder with a 1 ?m diameter, and it has a mass of . Its chromosome consists of a single double-stranded chain of DNA 700 times longer than its body length. The bacterium moves at a constant speed of 20 ?m/s, though not always in the same direction. Answer the following questions about E. coli using SI units (unless specifically requested otherwise) and correct significant figures. a. What is its length? b. Diameter? c. Mass? d. What is the length of its DNA, in millimeters? e. If the organism were to move along a straight path, how many meters would it travel in one day?
Lecture for Exam 3 Ch. 12—Membrane Transport 6. Explain in detail how the Na+/K+ pump works. - Primary active transporter, because it requires ATP to hydrolyze while it is transporting and both are going opposite of their electrochemical gradients. - Is also an Antiporter, because it has opposite physical directions (K+ going in, Na+ going out.) - Na+/K+ pump: transports 3 Na+ OUT and 2 K+ IN; both against the electrochemical gradient. - Charge difference across the plasma membrane: Na+/K+ pump (+1 net out), - molecules inside, K+ leak channels (always open and allows K+ to leak out.) Inside is more – than the outside = membrane potential. 7. Give 2 important functions of the Na+/K+ pump. - Na+/K+ pump: keeps cytosol [Na+] low & controls neuron activity states. 8. Describe the 2 types of glucose transporters. *Na+ Electrochemical gradient used by secondary transport - Na+/glucose symport: in apical membrane of epithelial intestinal cells. Active glucose transport from lumen into cytosol coupled with passive Na+ transport. Changes sides opening: extracellular side, then cytosol side Changes shape: dumping glucose inside epithelial cell from lumen, gradient dissipates over time as more sodium is - Basal Membrane uniport: passive glucose transport into bloodstream 9. Give functions of the H+ pump. *In just plant cells: - H+ (hydrogen ion/proton) pumps: to establish H+ gradient for secondary transport. Keeps higher concentration of H+ outside than inside; same reason as why animal cells keep high concentration of Na+. *In both animals and plants: - H+ (hydrogen ion/proton) pumps: to acidify organelles. 10. Explain how ion channels are selective. *Channels can only do passive transport and have to be able to tolerate charges inside the port & need side chains that are going to be of the opposite charge of what is being transported. Doesn’t have to be full negative, can be partially negative. - Ion channels have specificity: selective filters - R-groups form a pore: the diameter and charge select the ion type/ limit which ions can pass through. (Ex: K+ channel—C=O; O pulls/attracts cations; pore is too narrow for Ca2+ & is too wide for Na+.) 11. Describe the 3 main types of gating mechanisms for ion channels, and discuss examples of each. - Ion Channel Gating: 3 stimuli— Will be closed unless stimulus causes it to open. Voltage (VG): gate on channel will be opened or closed depending on the charge/voltage distribution across the membrane. Ligand (LG): have a binding site for a substance that will cause the gate to open or close; ligands that bind can come from the outside (if plasma membrane channel) or inside the cell. Mechanical/Stress/Stretch (MG): a physical stimulus/force that is put onto a part of the channel that causes the gate to open or close. 12. Describe in detail the formation and propagation of an action potential in a neuron. *Without ion channels, neurons would not work, because the signals (action potentials) depend on ion channels. - Neurons: form and carry signals from one part of your body to another part of your body. Action potential starts at Axon Hillock, travels down the axon, to the end of the axon terminal where signals are passed to another cell. Need ion channels to form action potentials and need them in the right places so that signal goes in the same direction every time. *Squids have large neurons and the axon is actually referred to as the “giant axon.” The axon carries signals to muscles. (Their neurons are about 1mm wide and 10cm long. These were the first studies of neurons in the 1930s.) Intracellular recording: electrodes in/out of the axon monitor membrane potential. Ions flowing in and out change the membrane potential. - Resting: resting membrane potential -20 to -200 mV = equilibrium potential of K+ (K+ leak channels open, K+ leaks out until reaching equilibrium as others come back in.) (Ex fig: Ion channels that are permeable to potassium (K+) are opened when the neuron is resting/not signaling/not forming an action potential. Potassium channels leak K+ out of the cell. Eventually, there will be an equilibrium.) - Nernst Equation: calculate the equilibrium potential of an ion V= RT/zF InCo/Ci R gas constant T temp in K z charge F Faraday’s constant Co/Ci concentration outside over concentration inside the cell (Ex: At 37C for K+ the equation simplifies to: V= 62logB.10 ([K+]0/[K+]i) If [K+]i= 140 mM and [K+]o = 5 mM, then V= 62(-1.447) = -89 mV “polarized”) *In most mammalian cells, the resting potential is around -70 (a little more positive) and the membrane is slightly more permeable than to other ions. - (Ex fig: Neuron depolarizing to the threshold at the axon hillock (-40 mV); Action Potential forms, then rapidly depolarizes (~+50 V), then rapidly repolarizes back to rest.) A strong pull for K+ to get into cell. Sodium channels are opened at threshold voltage are stimulated to open by a change in a membrane potential, gated by voltage (VG).) - (Ex fig: Whenever threshold VG potential is reached and channels open, sodium comes in making cell very positive inside. Voltage change exerts electric force on voltage sensor domain ((side chains of amino acids sensitive to charge changes around them and whill therefore change shape of protein in that area to allow ions to flow or be blocked.)) At +50, Sodium channels go to a state called “inactivated”, stop the influx of ions, but the channel has to go back to being closed and can then reopen again. This is the refractory period, where it can’t make an action potential during this state.) - (Ex fig: As Na+ enters, the membrane potential depolarizes. VG Na+ inactivate at about +50 mV for about 1 msec (and stops the influx of Na+). Channels return to the closed configuration as the membrane potential returns to rest. K+ leak channel are few in number and wouldn’t be efficient enough to bring back down in 1 msec. So…) - (Ex fig: Repolarization “delayed” VG K+ channels open as the VG Na+ channels inactivate. VG K+ and Leak K+ allow the efflux of K+. Slight hyperpolarization until the VG K+ close. There is now a high Na+ inside while a high K+ outside, which is not good, so Na+/K+ has to restore concentration gradients to what they need to be which is the resting concentration. Both forces are acting in the same direction to let K+ out so it is now a gush instead of a leak..) - (Ex fig: “All or None” Propagation: Action potential travels down the length of the axon without diminishing from the hillock to the terminal. How There are voltage gated Na+/K+ channels at (V1, V2, and V3) each one of the electrodes. When the Na+ channels open and Na+ pours into the axon, it depolarizes the membrane, diffuses into the cytosol in both directions, and reaches threshold. This happens at every action potential. By the time that it happens at the previous one, the only place that will form a new action potential will be the next one to the right, because the previous Na+ channels that opened are inactivated and cannot open again until after they close. So, it can only happen where the action potential has not been, forcing it to go forward only. Allows size of the action potential to always be the same. The action potential is, therefore, regenerated. IN SHORT: The cytosol carries the depolarization wave a short distance. Threshold opens and new Voltage Gated Na+ channels and new Action Potentials form. Inactivated Voltage Gated Na+ channels are now unresponsive and the Action Potential travels forward only (away from the hillock.) IF STIMULATED AT V2 TO THRESHOLD (in a pretend environment): the first place you will see an action potential is at V2 and then simultaneously at V1 and V3. This is because there are voltage gated channels along the axon, but in an abnormal environment and stimulation at a different place, Na+ diffuses in different directions and because the other action potentials have not occurred yet, they will be stimulated. 13. Give a detailed description of the events at the synapse. - Synapse: the axon terminal (of the presynaptic cell) relays signals to the target cell (the post- synaptic cell.) The usual way this happens is that the electrical signal that propagates down the axon to the terminal gets converted to a chemical signal that crosses a very small space called the synaptic cleft. The chemicals here are Neurotransmitters contained in the presynaptic vesicles. - (Ex fig: The release of NT’s at the terminal: the Action Potential opens VG Ca2+ channels. Ca+ influx causes Neurotransmitter Vesicle fusion with the presynaptic membrane. The Neurotransmitter is released into the cleft, diffuses, and binds to the receptor on the postsynaptic membrane. ) - (Ex fig: If the Neurotransmitter is a Ligand Gated ion channel: Neurotransmitter (ligand) binding opens the ion channel, creates an electric signal in the postsynaptic membrane. (IF channel for NA+, depolarize. IF channel for Cl, hyperpolarize.)) - (Ex fig: Acetylcholine ((Ach)) receptor at the neuromuscular junctions: the Receptor binds Acetylcholine, Na+ channel opens, Na+ influx, muscle cell depolarizes, Action Potential forms… Contraction.) - (Ex fig: Channels have a faster flow than transporters: rush of ions in/out forms electrical signal; rapid response. For example, a Venus Fly Trap.) - (Ex fig: Patch Clamp Recordings: records electrical currents through ion channels. Is sensitive and fast. Cell-attached patch: attach electrodes, has tight suction, and records channels under the electrode. Detached patch (cytoplasmic face exposed): can pull off that piece of membrane, record currents through those channels, but lets you change bath solution and expose the channels to different ions in the bath to determine what kinds of ions are permeable through the channels. Can use inhibitors to try to block channels, as well. *Faraday cage: protects readings from any unwanted electrical noise *Anti-vibration table: protects readings from any unwanted vibrations - (Ex fig: Cochlea: sound is converted to fluid waves, moves to the basilar membrane, and lifts the hair cells in the Organ of Corti. The bundle of Sterocilia on the hair cell bends when it contacts/rubs against the Tectorial Membrane. Stress gated/Stretch gated/Mechanically gated ((MG)) ion channels: occur at the Stereocilia tips. The Gates are tethered to the tip links, the Tip links pull open the gates, there is a cation influx, and the hair cells signals to the Auditory Nerve. Usher-deafness: at birth/progressive loss of hearing; a mutation in myosin, mutations in harmonin (between mysonin and tip-link of actin) Loud sound-induced deafness: when hair cells lose their tip-links, they can no longer transmit sound signals. Human hair cells tend to die when there is a loss of tip-links. *Sea anemones don’t have ears, but have hair cells in their tentacles. Patch-clamp recording image professor took. Bent the hair cell and recorded curves from every way the cells were bent. Sea anemones have hair cells that are being used in a similar way that our hair cells are being used (they also have mechanical-gated.) Ch. 5— DNA and Chromosomes 1. Describe the structure of DNA. - Sugar + Phosphate + Nitrogenous Base = Nucleotide Phosphodiester bonds are between sugar-phosphate groups. Hydrogen bonds are between the base pairs (A:T 2 bonds between; C:G 3 bonds between) - DNA: a nucleotide polymer Strand: 4 types of nucleotides that are covalently bonded in a linear array with the 5’ and 3’ ends (numbers are from the carbons and sugars) DNA double-stranded helix: each strand is a sugar-phosphate backbone with bases on the inside. Both strands are complementary to eachother, not identical & are also anti- parallel. The width is about 2 nm wide, is right-handed (twisted towards the right), has 10 nitrogenous bases per turn, and has Major (larger spaces on one side) and Minor (smaller spaces on the other side) grooves. (Naked, stretched-out DNA is skinnier than actin filaments) - (Ex fig: Information: a linear sequences of bases; the Language are 3-letter words, each coding for a particular amino acid.) - (Ex fig: Chromosomes: DNA partitioned into long molecules; each chromosome has a linear array of genes. Genes: code for polypeptides or RNA. *The longer the chromosome, the more genes it contains.) 2. Discuss the general features of eukaryotic chromosomes. - Should have 46 chromosomes in human tissue; 23 pairs of chromosomes that are similar to eachother. Pairs originally came from two people but united to make one pair (for offspring). 3. How is karyotyping done - Karyotype: array of mitotic chromosomes “painted” or stained with chemicals. detect chromosomal abnormalities. “Painting”: DNA hybridization to fluorescent cDNA probes. Somatic cells: 2 versions of each chromosome, or “homologues” *X- much larger and can carry more genes (x-linked example is red/green color blindness) Y- smaller and can carry less genes *Chromosome 21 is smaller when one has Down Syndrome and have an extra (third) chromosome. If you have an extra 21, the effect would be less severe, because it is so small, and allows those affected to live. *Translocation: when a piece of one chromosome is moved to another. - (Ex fig: Extra/missing pieces ((ex: ataxia, 12 has a piece of 4 attached to it.)) Extra/missing chromosomes: ((ex: Down Syndrome 21 x 3, Turner XO in females and makes infertile & secondary sex characteristics don’t develop, Klinefelter XXY trisomy produces a male that has an extra X and is similar to Turner’s in which the male has physical problems including not being able to reproduce as well & secondary sex characteristics don’t develop.) 4. Describe the changes in chromosomes during the cell’s life cycle. *If not looking at cell in mitosis, chromosomes are very long and skinny. In mitosis, the chromosomes condense and are shorter, fatter, and easier to see. - Interphase: chromosomes change shape (replication/transcription); chromosomes are decondensed and allow proteins the access to the double helix; (chromosomes do still exist in the interphase nucleus!) - Mitosis: partition/separating the copies of its DNA to opposite/daughter cells/new nuclei; chromosomes are condensed and copies are moved by the mitotic spindle (made of microtubules) to the opposite poles. *(Ex fig: Sister Chromatids: (identical sister) pieces of chromosome stuck together by a centromere; a replicated/condensed chromosome = 2 chromatids attached at a centromere.) 5. List the levels of DNA packaging and the resulting condensation achieved by each - Naked DNA (2 nm wide) - Chromatin: DNA with proteins, together. - Interphase Chromatin: Nucleosome: DNA wrapped 2x around a histone core (11 nm wide). Nucleosomes (individual beads) are coiled into fiber (30 nm wide; called the “30 nm fiber”). - Mitotic Chromatin: Condensation: fibers loop (300 nm), twist together (700 nm), and supercoil (1400 nm). - (Ex fig: light circles surrounded by dark ((nucleosomes surrounded by DNA)); if expose DNA to denaturing, exposes beads ((nucleosomes)) with DNA wrapped around it.) - (Ex fig: nuclease breaks phosphodiester bonds; nuclease will have access to break the bonds where the linker DNA is in between nucleosomes; nuclease can digest DNA between. Can further process what nucleosomes are. Dissociated with salt, a piece of DNA that is 147 base pairs ((always the same length)), histone core is always the same and show that the DNA is wrapped twice around the nucleosome. The histone proteins have an abundance of positively charged ((base)) side chains which are on the outside of the core, because DNA is negatively charged. When breaking the core, separates into four types of polypeptides ((8 total that make up the core—octamer)). Nucleosome Core: 8 histones with 4 types; 2 of each.) *Proteinase K (in lab)- digest proteins; a hydrolase that breaks peptide bonds between amino acids. - (Ex fig: Histone 1 has two tails with a triangular-shaped head, with C and N-terminuses. Guides DNA as it comes off of the Histone via the shape that makes it bend to direct toward coiling. Histone 1: linker histone that helps to form 30 nm fiber. The globular region bends DNA that is coming off of the core while the tails hold DNA onto the core.) - (Ex fig: Tails poking out from the Histone core; are places where modifications, covalently attached groups can be attached to the tails to tag and be read by another enzyme. Types of covalent modifications are important in controlling how condensed that piece of Chromatin is. Core histone tails: protrude and are covalently modified.) - (Ex fig: H3 tail modification: methylation, acetylation, and phosphorylation. Not all modifications happen at the same time, they are just possibilities. Heterochromatin formation (pattern affects gene transcription ((silenced or expressed)): Methylated: very tight chromatin; gene will not be able to be transcribed, because it is too tightly wound for transcription to take place by RNA polymerase. Cannot get in to do transcribing. Causes gene silencing. Acetylated: expression of gene, because the chromatin is less condensed and allows RNA Polymerase in. Acetylated and other is Phosphorylated: expression of gene, because the chromatin is less condensed and allows RNA Polymerase in. - Remodeling Complexes (CRCs): pattern is read by Chromatin; will keep tightly wound or loosen that part of DNA. Requires ATP (Hydrolysis) and slides DNA along the histone core until they move away from the DNA if the gene needs to be expressed. Will move the other way to wind tighter if needed to be silenced. Allows proteins to have access to DNA for transcription, replications, and repair.) 6. Distinguish between heterochromatin and euchromatin. - Interphase: there is a mix of (~10%) Heterochromatin: highly condensed form that occurs in cells; areas of the DNA where there is gene silencing/no genes present. Mostly centromere and telomeres. (~90%)Euchromatin: loosely packaged/less condensed chromatin *Nucleolus is not heterochromatin; it is where rRNA and ribosomal subunits are made. - Long-lasting heterochromatin: inactive genes; normally active genes are misplaced in heterochromatin and may lead to disease (severe anemia from Beta globin silencing) - (Ex fig: one of two X’s is converted to heterochromosome during. This is a random event in which some will super-condenses the paternal or maternal chromosome,. But once it happens, it will divide and give rise to daughter cells and converts the same chromosome to while the other is doing the opposite. All cells after will produce the same one. - Extreme Heterochromatin: X inactivation in female mammals. - Barr Body: one X inactivated/cell X: condensed randomly in early embryo; all descendants of the cell inactivate the same homologue. Epigenetic (beyond gene sequence) inheritance. Xi: high DNA methyl, low histone acetyl, low H3 K-4 methyl, high H3 K-9 methyl. - Black & Orange fur carried by X-chromosomes in calico female cats. Chromosome of each type, one gets randomly changed to heterochromatin and forever gives rise to cells that give the opposite color fur. The daughter cells change the same one each time. The white is controlled on a different set all together. This is an X-linked trait only seen in females, because the males only have one type. Ch. 6— DNA Replication, Repair, and Recombination 1. Explain the experiments that provided evidence for semiconservative DNA replication. - DNA Replication: two strands separate to be replicated and each strand serves as a template for a new strand. Each molecule is partly old and partly new= semi-conservative. The original DNA molecule is anti-parallel to the template strand and, likewise, the daughter DNA molecule is anti-parallel to template strand as well. Semi-conservative: each pDNA (parental) is a template for building a dDNA (daughter) strand. Conservative: pDNA (old) intact, new DNA= 2 dDNA (new) strands Dispersive: each strand = pieces of pDNA (old) and dDNA (new) - Meselson and Stahl (grad students) experiment: 1) Grow 2 bacteria cultures; one with “heavy” isotope of nitrogen N15 and another with “light” isotope of nitrogen N14. *Bacteria can grow a new generation in only 20 mins. *Solution they are grown in is called the “culture medium.” *Isotopes are different in neutrons. The Nitrogen is used for forming dDNA. *Took test tubes they filled with Cesium gradient, put DNA on top of it, and ran the centrifuge. DNA (extraction) will be pulled through the gradient until it reaches a gradient that matches its own. RESULTS: N14 culture = light DNA, N15 culture= heavy DNA. 2) Transfer heavy bacteria to light medium; extract DNA after 1 generation. RESULTS: DNA molecule (both strands) was all intermediate in density/weight. Was a combination of both heavy and light strands. (Semi-conservative or dispersive Have to separate the two strands from the molecules and run the single-stranded DNA. If semi- conservative, one band would be heavy and one band would be light. If dispersive, it would be intermediate.) 3) Heat DNA to separate the strands. RESULTS: 2 bands; 1 heavy and 1 light (semi-conservative!) 2. Describe the replication origin. - At origin, there will be a bunch of proteins that bind to DNA that form a bubble and once that is formed, can get new DNA. DNA will be replicated in both directions. Each one of these is called the replication bubble. - Replication origin: DNA sequence recognized by the initiator proteins (IPs). Don’t all start at the same time. - IPs create the replication bubble; proteins access to template DNA - Each bubble = 2 opposing forks where DNA is replicated. *Over time, replication bubble gets larger/grow. - (Ex fig: forks move 100nt/sec (humans) and the Bubbles grow to replicate the entire chromosome. The cell gives/splits histones between old and new DNA molecules and tags get passed onto daughter chromosomes. ) 3. What is the fundamental reaction by which DNA is synthesized - DNA Synthesis: condensation (anabolic) reaction Phosphodiester bond attaches new nucleotide to 3’ end and is catalyzed/coupled by the DNA polymerase enzyme (can only add to a 5’ end). dNTP (deoxy-nucleoside triphosphate) hydrolysis: the energy for this reaction that breaks a phosphoanhydride bond. 4. Why is synthesis at the replication fork asymmetrical - DNA Polymerase: can only synthesize from the 5’ to the 3’ (reads template 3’ to 5’) and pDNA strands are antiparallel. - Compared to the fork movement: Leading strand: one new strand made in the same direction; made continuously; 2 in each bubble Lagging strand: the other made in the opposite direction; made discontinuously in segments (Okasaki fragments); 2 in each bubble 5. Can DNA polymerases synthesize a new DNA strand “de novo” (from the beginning) - DNA polymerase cannot synthesize “de novo” or “from the beginning” - DNA primase: lays down the primer (a short piece of RNA—has U instead of T and has a ribose instead of deoxyribose). Needed for DNA polymerase to attach nucleotides to. Are removed/replaced with DNA (repair polymerase) Lagging strand: primer must be added before each Okasaki fragment; as many as the Okasaki fragments Leading strand: only need one - Ligase: seals the gap between the 2 new DNA pieces 6. Describe the molecules involved in synthesizing the leading strand versus the lagging strand - Leading and Lagging: DNA helicase: responsible for moving replication fork; unwinds pDNA double helix and separates 2 strands from eachother Single-strand DNA-binding protein: binding to single-stranded piece and stabilizing it; prevents from recoiling back Sliding Clamp: holds polymerase onto the template strand Topoisomerase: relieves supercoiling ahead of the fork - Lagging strand: SSS Proteins: stabilize ssDNA Enzyme complex: replication machine; moves along pDNA at the replication fork - Topoisomerase ahead of fork - Leading Strand: Helicase, Primase-once, Polymerase & Sliding Clamp, Ligase-once - Lagging Strand: Helicase, Single-strand stabilizing proteins (SSS), Primase-each Okasaki fragment, Polymerase & Sliding Clamp- multiple Okasaki fragment, Ligase-each Okasaki fragment 7. Explain the role of telomeres and telomerase. 8. Describe DNA repair mechanisms. 9. Discuss ways that DNA mechanisms may change despite repair mechanisms. 10. Describe mobile genetic elements. Ch. 16— Cell Communication 1. What are the basic steps of cell signaling 2. Define the different types of signaling mechanisms and cite an example of each. 3. How do cells within multicellular organisms respond specifically to external signals 4. Distinguish between slow and fast responses to signals. 5. Describe 2 examples of extracellular signals using intracellular receptors. 6. List 5 ways in which cells benefit from signaling cascades. 7. Discuss 3 types of cell surface receptors with examples.