Biology Exam #2

Frederik Griffith Experiment

1928 British bacteriologist Studying bacteria that cause pneumonia, streptococcus pneumonia Two different strains studied: rough, which is the R strain (only has peptidoglycans surround bacteria as part of cell wall); other is smooth strain (S strain), has capsule (along with peptidoglycans) that surrounded the bacteria that composed of a slippery polysaccharide coat This capsule allowed the bacteria to invade the host's immune system and therefore allowed them to be virulent and eventually, to kill the host Injected various strains into mice and wanted to see if mice would live or die—R strain alone (lived, thus proving R-strain is benign) and S strain alone (died, polysaccharide coat allowed to kill host) Then injected heat-killed S strain and the mice survived (because denatured the lethal properties) Also injected heat-killed S strain of bacteria and mixed it into a test tube with live R-strain (mice ended up dying = TRANSFORMATION) Live R-strain cells were transformed into the virulent S-strain cells (absorbed genetic material of the S-strain to become lethal) Some molecule in bacteria that gives them the ability to become pathogenic (this molecule can be transferred between bacteria in a process called TRANSFORMATION) But did not know what the transforming agent was: protein? Nucleic acids?

Avery, MacLeod, McCarty Experiment

1944 Continue Griffith's experiment, but this time, they did some manipulation which allowed them to determine what was the transforming factor Started with heat killed streptococcus cells, which contain different types of macromolecules, and by chemical means, removed the lipids and the carbohydrates Then, divided these cells into three different populations and added particular enzymes to digest one specific type of macromolecule in each of the treatments One treatment, added protease, which digest the proteins Second treatment, added RNase, which digest the RNA Third treatment, added DNase, which digest the DNA Took these digested, heat-killed S-strain bacteria and added each treatment to a mixture of our cells, injected them into the mice, and asked if the mice would be able to live Removed protein with proteases, the mixture of bacteria was able to kill the mice and transformation did take place (suggested proteins NOT transforming factor) Similar results with the cell treated with RNases, cells also able to transform the R-strain into the S-strain (thus the RNA that was digested was not the transforming factor) Heat-killed S-strain treated with DNA =NO transformation when remove DNA, thus DNA must be transforming factor present in the S-strain of cells, which was able to transform the R-strain Controversy continued (even though figured out transforming molecule is DNA)

Chase and Hershey Experiment

1952 Controversy put to rest Studied the T2 bacteriophage, which is just a virus that infects bacteria When infects bacteria, it can produce more copies of itself and eventually kill the bacteria Life cycle of bacteriophage is shown When virus lands on bacterial cell wall, it can inject its genes into the bacteria Those genes then put all of the genetic material necessary for production of new virus particle, and when assembled in the bacteria, can erupt from inside the bacteria in process known as lysis, and go on and infect other bacteria Important to design of experiment that the virus is capsid (which is what initially contains the genetic material) will remain outside of the cell, while the genes necessary to encode for the production of new viruses will be injected into the bacterial cells (viral capsids will float away after genes have been injected into bacteria) Labeled the viruses with either radioactive P32 (isotope of phosphorous) or radioactive S35 (isotope of sulfur, which is a key component of proteins--found in cysteine and thiamine) and phosphorous is key component of nucleic acids, or in this case, viral DNA Labeled with radioactive isotopes and then infected e coli bacteria with these viruses Then agitate the cultures by putting the bacteria and viruses that infected them into a blender, the viral capsids (which remained outside of bacterial cells) will dissociate and float away into the solution, and when they then centrifuge the solution (spun them at very high speeds), they could pellet the bacteria cells at the bottom of the tubes Because genes had infected the bacteria, they would be found in the pellet along with the bacteria while the viral capsids (which is the protein coat) which had floated away and be found in the solution Two competing hypotheses they were testing: DNA hypothesis: The radioactive DNA, if it was actually the genetic material, will be located within the pellet after centrifugation Protein hypothesis: The radioactive protein, if it was actually the genetic material, will be located within the pellet after centrifugation Results: Radioactive DNA (P32) was found in the pellet following centrifugation Radioactive protein (S35) was found in the solution following centrifugation Concluded that in fact viral genes of T2 bacteriophages did consist of DNA, while the viral coats consist of the protein Consistent with Avery's experiment, which suggested that transforming material with streptococcus bacteria, or the genetic material of the genes in the bacteriophage, was in fact, DNA DNA was finally accepted as the genetic material

Howard and Pelc Experiment

1953 Founding out when during eukaryotic cell division DNA synthesis occurs Use a pulse-chase experiment Feed radioactive thymidine (deoxynucleoside-- a sugar and thymine base without the phosphate) to cells growing in a culture (pulse) After 30 minutes, washed any unincorporated thymidine out of the cell cultures and chased with a lot of nonradioactive thymidine Then visual radioactivity-- looking for where thymidine is incorporated into DNA during the cell cycle-- Exposed these pulse chase cells immediately to photographic emulsion -- dark spots where nuclei that had incorporated the radioactive thymidine...found that they were incorporated in the S phase (also shows the cells that entered into mitosis did not have radioactive thymidine yet) Thus, DNA synthesis occurs in S phase, or interphase, of the cell cycle DNA synthesis cannot occur at the same time that the cell division is actually taken place (S phase and M phase are separate processes) How long does S-phase last? Feed radioactive thymidine to cells growing in the culture and only the cells synthesizing DNA (in S phase) will incorporate the radioactive label (pulse) After 30 minutes, wash unincorporated radioactive thymidine out of cell culture (chase) Spread out cells and exposed them to photographic emulsion, but this time, they did not do it immediately--instead, they waited for certain time frames after the chase (2, 4,6..16 hours after) The cells are growing asynchronously -- cells are spread out all around the cell cycle-- some cells actively dividing in mitosis during pulse or could be in S phase or intermediates (G phases) The time periods determine the number/percentage of cells in mitosis that have radioactive labels Cell cycle is clockwise Any cells in S phase during pulse are going to be labeled...and then going to track the radioactive cells through the cell cycle as time passes to see which ones (chromosomes in cells) are undergoing mitosis 0-4 hours after chase, no M-phase cells with radioactivity 4 hours, radioactive cells beginning to enter M-phase 4-12 hours after chase, show the amount of cells with radioactivity in M-phase (show the length of S-phase...some of the cells were at the end of S-phase, and some were at the beginning when fed with the pulse) 12 hours...radioactively labeled cells are leaving M-phase and going on in the cell cycle 12-20 hours, no radioactively labeled cells in M-phase (because have left to the rest of the cell cycle) At 24 hours, start to see the radioactively labeled cells enter M-phase once again S-phase was thus determined to last 8 hours Time between S phase and mitosis = 4 hours..this is the gap phase (G2) Time between mitosis and S phase = G1 gap phase...preparing to synthesize the DNA (also 4 hours?? looks like longer than G2 phase) A lot going on during gap phases, just cannot see it

Franklin, Crick, Watson and Wilkins Experiment

1953 Proposed a structural model for double-stranded DNA Franklin and Wilkins were using a technique known as X-ray crystallography to deduce the structure of DNA, this technique entails an X-ray beam and shining it on a crystal of the molecule who's 3-D structure you're trying to figure out--in this case, DNA Crystallized DNA would diffract the X-rays so that they could be detected on the photographic plate, and depending on the orientation of the molecules within that crystal, you can deduce from the diffraction pattern what the 3-D structure may be Deduced from this crystal structure that the 3-D structure of DNA was likely to be a double helix

The Meselson-Stahl Experiment

1958 How is DNA synthesized? Wanted to distinguish between the three different models (conservative, semi-conservative and dispersive) for how DNA might be copied They separated parental and newly-synthesized DNA on the basis of its density to figure out the model of DNA synthesis Used heavy isotopes of nitrogen (nitrogen because of the nitrogenous bases which will incorporate the isotopes in them)-- N15 is the heavy isotope and N14 is the regular isotope Grew the bacterial cells in a growth medium of only the heavy nitrogen isotopes (N15) as sole source of nitrogen for the bases for many generations --starting population of bacterial cells then, would only have DNA made up of the heavy nitrogen isotope = generation zero DNA sample Then, transferred these same bacterial cells to a medium containing the normal nitrogen isotope (N14) and allowed those cells to divide once -- then took these divided once bacterial cells out and purified the DNA from them = generation one DNA sample Then allowed the bacteria to divide a second time in the normal nitrogen isotope (N14) medium, and collected a sample of the divided twice bacterial cells and purified the DNA from them = generation two DNA sample Then they centrifuged these three sample of DNA, separately, ran through something known as a cesium chloride gradient (just salt that has different densities within its test tubes)...could separate out the DNA and now distinguish between all the new strands The heavy nitrogen, when DNA is subjugated to centrifugation, migrates further through the tube because its heavier The normal (lighter) nitrogen does not migrate/ centrifuge as far in the tube because lighter Generation zero = the original "parental" DNA (with a heavy nitrogen isotope) Generation one = Semi-conservative and Dispersive models have DNA helices that are hybrids or intermediates where as the conservative model shows two different DNA helices, one containing all heavy nitrogen and the other containing all normal (lighter) nitrogen Generation two = Now can differentiate between the semi-conservative and dispersive models because after two generations in semi-conservative, expect that half of the double helices should by completely low density DNA and the other half will be hybrids or intermediates whereas dispersive shows that they are all still hybrids or intermediate density DNA Results: Whichever model correlated with the results of the centrifuge tube in the cesium chloride gradient would be deemed correct....as determined by the DNA in the centrifuge tube, generation zero was completely high density, at generation one it was completely hybrid (getting rid of conservative as an option), and at generation two, half the DNA was low density DNA and the other half was intermediate density DNA, thus the results were only consistent with semi-conservative replication, in which each parental strand is used to synthesize a complementary strand Top of centrifuge = lower density Bottom of centrifuge = higher density

Xeroderma pigmentosum (XP)

A disease characterized by sun sensitivity and skin cancer-- occurs in people with defects in the nucleotide excision repair protein development Cells from XP patients just can't survive to even intermediate doses of UV light Cultured cells from XP patients are extremely sensitive to UV light and cannot repair DNA damage after UV exposure Why do XP patients develop cancer? -- Some residual repair of UV damaged DNA even in XP patients and this repair is very air-prone...so DNA replication across the damaged DNA can result in mistake that become permanent mutations which leads to cancer in XP patients

Q

Also known as CoQ and Ubiquinone Small lipid soluble molecule that acts as a kind of convergence point in the chain so that electrons sent both from NADH and FADH2 will eventually arrive here and then Q transfers to the rest of the chain Only protein that can bring the electrons from Complex 1 and 2 to Complex 3 The mediator at the convergence point

Rubinstein-Taybi Syndrome (RTS)

An example of a human disease caused by defects in chromatin modifying enzymes Very rare-- characterized by broad thumbs, big toes, slow growth and upper airway infections It is caused by mutations in genes named CBP or p300 because these genes code for HATs -- thus chromatin structure is not being regulated correctly During human development, certain parts of chromosomes need to be condensed so that the genes are not being expressed, and other times, chromosome need to be decondensed so that gene expression can occur In these patients, the regulation of the condense/decondense of chromatin is not right and thus, certain genes may be expressed when they are not supposed to, thus causing these developmental abnormalities

What happens when mistakes are made during DNA replication?

Based on the physical chemistry of the hydrogen bond, one would predict an error rate of 1 in every 10,000 - 100,000 bases polymerize --unacceptable! so what prevents the mistakes?? Two different lines of defenses that take care of the mistakes-- These two can be fixed during S-phase -- 1.) Some DNA polymerases have proofreading ability -- Has a "backspace key" -- if a mismatched base is added to the end of the growing chain, many of the polymerases (including I and III) can detect that mistake and correct it; DNA polymerases are shaped kind of like hands--active site is found within the palm domain of the hand-- if mispaired last base pair at the end of this newly synthesized strand...going to destabilize the active site within the enzyme, such that this strand will move down to the polymerase where it is now at the exonuclease active site--this exonuclease acts to remove the deoxynucleoside monophosphates from the growing chain (going to remove that last base)...once that happens, the strand can then move back to the polymerase active site in the palm domain 2.) Mismatch repair -- Little bit slower than proofreading but still happens in S phase; mark the old strands of DNA with methyl groups which are added by an enzyme, but takes a little while for the enzyme to add the methyl groups to the newly synthesized strand...so thus...for a couple minutes, can detect difference between old and new strand...and whatever is on the old strand is presumed to be the correct base, so removes the mismatched base on the newly synthesized strand 3 proteins used for mismatch repair-- MutS is the protein that finds the mismatch and then binds to it...then going to peak/kink the DNA which causes other proteins to come on site, MutL and MutH MutH is the business end of the complex--job is to make a nick on the newly synthesize strand (knows which one to do it on because is not methylated) Then exonuclease can come and removes lots of different nucleotides there...then DNA polymerase I comes and fills in gap with appropriate base pairs to fix the problem Find mistake, get rid of it, replace/resynthesize Other lines of defense: Repair processes that happen outside of S phase Ex.) DNA damage caused by UV light (thymine dimers are created -- covalent bonds form between two adjacent thymine bases which will change the shape of the DNA and cause kinks and deformities) How are thymine dimers repaired? Repaired by nucleotide excision repair proteins --they recognize damage, remove it (exonucleases), and resynthesize and link (DNA polymerase I and DNA ligase)

Applications of PCR

Basically, any situation where you have very little DNA and need to make more copies for further analysis: Detection of mutations (screening newborns for inherited disorders) Bioterrorism prevention (maybe anthrax bacteria) Prenatal sex determination Screening for genetically modified foods Forensics Paternity testing All of these applications require more DNA than you have in the start, and that is why you need to do PCR Example -- Svante Paabo and colleagues --use PCR to amplify a short region of DNA from a neanderthal and compare it to modern humans to see if they had interbred when they had coexisted --compared DNA sequences and found that non-African populations share 1-4% of their DNA with Neanderthals...this was only made possible because they were able to sequence the entire genome

The Telomere clock hypothesis

Calvin Harvey showed that telomere shortening can cause cells to exit the cell cycle, enter into G0, and become senescent Cells with initially longer telomeres can undergo more cell divisions once they are put into culture (creates an increase linear relationship) Also, as we get older, our telomeres are getting older

Checkpoints

Can stop the cells from progressing inappropriately through the cell cycle

S phase and G2 phase

Chromosome are replicated, and each chromosome consists of two sister chromatids (a sister chromatid is a copy of a chromosome after S phase has been completed)

G1 phase

Chromosomes are unreplicated (shown as partially condensed just to make them visible...but not actually condensed at all)

Prophase

Chromosomes condense during this first phase of mitosis The early mitotic spindle begins to form

Krebs Cycle

Citric Acid Cycle (because first compound is citrate/ citric acid) Tricarboxylic acid cycle (because we have three acid groups on the first compound in the cycle (citric acid)) Once pyruvate is brought inside the matrix by transport proteins, we will oxidize the pyruvate We oxidize pyruvate by ripping of one carbon in the form of CO2 from the pyruvate and we will also reduce NAD+ to generate reduced form of that electron carrier, NADH (=redox reaction) Remaining two carbon of pyruvate will then be attached to carrier molecule, Acetyl CoA (coA carries acetyl group by a high energy bond involving a sulfur...) and that (the acetyl group binder to coenzyme A) will then carry the two carbon compound into the Krebs cycle First step: Attach acetyl group (of the two carbon compound) to the compound oxaloacetate, which then starts the Krebs cycle because attaching the two carbon compound to this four carbon compound to regenerate a six carbon compound called citrate Start with pyruvate...which will be broken down to release CO2 to make acetyl coA...later will generate two more molecules of CO2 in Krebs cycle (broken down all of the carbon-carbon bonds, now have mutilated glucose and left with CO2) In process of all of that oxidation, going to generate a little bit of energy in the form of ATP and also generate a lot of reducing power in the form of the reduced electron carriers , NADH and FADH2 **EACH STEP IN CYCLE IS AN OXIDATION-- looking to oxidize all intermediate to generate the reduced electron carriers...if the step is going in forward direction, carbon compounds being oxidized to reduce the electron carriers Succinate is being oxidized to generate fumarate...oxidized because removing some of those hydrogens and generating a double bond between two carbons and no longer fully saturated (Be able to look at the compounds and know which one is oxidized and which is reduced...but don't need to memorize names or structures of the compounds) Why a cycle? Because we start by using oxaloacetate as first substrate....but by end, have to regenerate oxaloacetate-- Can start with one of the products of an intermediate step in the Krebs cycle ...and end up with new oxaloacetate that then will take a pyruvate and begin a cycle of its own--shows that the process is a cycle (ALL comes back to oxaloacetate) Cycle means that adding any individual components will increase the speed of using up that pyruvate Carbons from pyruvate (attached to acetyl coA) in the first cycle are not shed; they are not shed until the second cycle when they are released as CO2 (instead, can find them in each of the intermediate compounds) Generate a lot of reducing power in this cycle, which will ultimately give us more ATP for this cycle Krebs cycle is also regulated by high concentrations of ATP and NADH that will block this pathway (just like in glycolysis)= feedback inhibition All of the reduced electron carriers will feed into electron transport and oxidative phosphorylation...which will generate all of the ATP As NADH is produced, huge amount of energy is stored within this electron carrier (much more than what is stored in ATP) -- so will power a lot of ATP generation when we get to the oxidative phosphorylation cycle (about 30 ATP for every one molecule of glucose) If no oxygen present, won't even start using pyruvate to generate any of that acetyl coA (also, there will be a build up of NADH because no oxygen to bring the electrons to, to regenerate NAD+...which then stops the pyruvate from entering the Krebs cycle)...so needs a way to bail out = Fermentation (30 ATP for every 15 glucose)

Principle components of DNA and RNA

Composed of nucleotide building blocks which has phosphate group, 5 carbon sugar and nitrogenous bases Nucleoside: just the sugar plus the base without the phosphate group The phosphate group has two negative charges on two of the oxygen atoms, which means there is a lot of negative charge to the side of DNA double helix which would tend to repel the two strands apart if it were to for the hydrogen bonds and hydrophobic interaction of the interior (which allows it to stabilize) --thus DNA and RNA have large negative charge due to phosphate groups Carbons in sugars are numbered with primes RNA has a hydroxyl group on the 2 prime carbon whereas DNA has a hydrogen atom on the 2 prime carbon RNA has a ribose sugar whereas DNA has a deoxyribose sugar Two kinds of nitrogenous bases: pyrimidines which are one ring bases-- cytosine, uracil and thymine (Uracil is only found in RNA and thymine is only found in DNA) purines which are two ring bases-- guanine and adenine A single nucleic acid strand has polarity (which means it can be read in a particular direction) -- have a 5' end and a 3' end The 5' end is where the 5' carbon sugar is bound to a free phosphate group The 3' end is where the 3' carbon of the sugar has a free hydroxyl group Purine-pyrimidine pair of nitrogenous bases that is the perfect shape to fit into the double helix Purine-purine is too much space whereas pyrimidine-pyrumindine does not fill enough space (neither are energetically favorable) The purine-pyrimidine pairs can either by cytosine/guanine, which will form three hydrogen bonds between the two bases or adenine/thymine, which will form two hydrogen bonds between the two bases (more hydrogen bonds, more stable) G-C base pair is more stable than A-T base pair which is more stable than A-U (RNA) base pair

Condensed v. Decondensed Chromatin

Condensed chromatin--nucleosomes tightly packaged with one another Decondensed chromatin-- places between nucleosomes where there is only DNA, and thus very open to enzymatic attack by things that might want to break the phosphodiester bonds, such as DNase I --will cut phosphodiester bonds and you'll end up with lots of little pieces of degraded DNA and released nucleosomes Can detect that the parts of chromosome that could not be digested were very tightly wound and compacted (thus condensed chromatin)

Chromosomes and the cell cycle

Contiguous stretch of chromatin Only visible during a brief time in the life of a cell (the cell cycle), when they condense, align themselves, and are separated into two new cells-- mitosis "Mito"- thread "Osis" - state of Interphase of the cell cycle: G1 phase -- cells have chromosomes in the nucleus and are unreplicated but partially condensed to make them visible S phase -- chromosomes are replicated and become composed of two different sister chromatids (where DNA synthesis occurs) G2 phase -- the cells prepare for cell division M phase or mitosis -- At start, replicated chromosomes condense and then the sister chromatids eventually separate. Two daughter cells are formed by cytokinesis-- Mitotic spindle is what drives mitosis--connects to chromosomes and drives them to opposite sides of the cell--composed of microtubules (which are made up of alpha and beta tubulin dimers that make up a hollow tube-like structure that has a plus and minus end); plus ender where microtubules generally tend to grow faster--microtubules originate in centrosomes of animals, which are duplicated during G1 and S phase of the cell cycle (composed of two different bodies known as centrioles) Chromosomes MUST be copied before cells can divide = replication (once copied, then need to be condensed)

Chemical uncoupler

DNP--a small lipid soluble molecule--if injected, it will fit right into the inner membrane and carry protons from intermembrane space back to matrix Dissipate gradient without any energy--creates a "back door" for the protons; letting all chemical energy stored in protons leak back into matrix with out the conversion to mechanical energy and the formation of ATP (burn glucose and instead of storing the energy in the form of ATP, just making heat)--why it is a good weight-loss mechanism

Fermentation

Does not require oxygen Allows us to reoxidize NADH to NAD+, then we can break down another glucose molecule to get a little bit more ATP Reduce pyruvate to produce lactate (in humans) -- done without intermediates because pyruvate accepts the electrons from NADH Production of lactate in humans from fermentation; LDH enzyme is used to produce lactate molecules from the pyruvate to regenerate that oxidized NAD+ In yeast, we see alcohol fermentation: Product will be ethanol, because use the enzyme ADH to reduce pyruvate as the bailout pathway (also peel off carbon atom as CO2) to regenerate the oxidized NAD+

DNA structure

Double helix Looking specifically at D-form DNA (other forms depending on conditions...but this is most common form in cells) Have deoxyribose sugars (pentose sugars) as well as phosphorus atoms facing outwards--this forms a sugar-phosphate backbone Nitrogenous bases pair off and face inwards in the helix D form DNA exists as a right-handed helix And two secondary structural attributes of DNA -- major groove and minor groove -- important because the major groove has a lot of space for other molecules within the cell to recognize the particular base pairs present in that stretch of DNA (transcription factors are the proteins that recognize the information within the major groove; they can make hydrogen bonds with some of the bases, which allows transcription factors to carry out their job during transcription) Interactions that promote the stability of the secondary structure of DNA double helix-- hydrogen bonds between the nitrogenous bases; hydrophobic interactions between the nitrogenous bases as well (BASE-STACKING which excludes water from the space in-between the bases, which is favored energetically) Base pairs are not oriented directly in the middle of the helix, actually align a bit to the side--this misalignment causes the major and minor groove to form (not a completely symmetrical helix)

Number of chromosomes per cell

During S-phase, number of chromosomes of cell do not increase...but number of chromatids and DNA molecules does increase (will double) During anaphase is when the number of chromosomes double due to the connection at the centromere being broken--where sister chromatids become chromosomes (and DNA and chromatids are still the same amount as during S-phase, G2, as well as the other prior stages of mitosis)

Telomerase

Expressed in our germline cells (sperm and eggs) but is turned off in most somatic cells Therefore, it is thought that most somatic cells have a limited number of divisions that they can undergo So, could aging be slowed down if the "telomere clock" can be turned off?? -- continually express telomerase so that DNA synthesis and thus cell replication continues....problem--cancer! Cancer cells often reacquire the ability to produce telomerase -- this is one reason why cancer cells are considered "immortal"

FUCCI

Fluorescence ubiquitination cell cycle indicator (FUCCI) A direct way to visualize the cell cycle Utilizes our knowledge that levels of cell cycle regulatory proteins rise and fall during cell division One protein fused to red fluorescent protein, another protein fused to green fluorescent protein Can monitor the position of the cell cycle that each cell is in G1 -- concentration of G1 protein will appear red S phase and G2 -- concentration of G2 (and S) protein will appear green (M phase as well) In-between G1 and S -- will appear yellow In-between M phase and G1 -- will appear colorless

How do we visualize the products of a PCR reaction?

Gel electrophoresis Separates DNA molecules of different sizes, as they move in an electric field through a gel-like matrix (do not have to worry about charge because DNA is already negative due to phosphate groups--so do not need SDS page like in proteins to neutralize charge) Goes from negative to positive end Use a gel made out of agarose (a substance extracted from seaweed) Smaller molecules will move faster Process: 1.) Isolate DNA and pipette the DNA into wells of an agarose gel 2.) Put the gel in a chamber with a salty solution. Expose the gel to an electric current. DNA (negatively charged) will migrate through the gel away from the negative electrode towards the positive electrode 3.) Stain the gel with a molecule that binds tightly to DNA (ethidium bromide) 4.) Visualize/take a picture-- Ladder all the way to the left will show known sizes/lengths of DNA to compare to the size of the pieces of DNA we are testing in PCR

Cytochrome

Has a "heme" or prosthetic group, which means the part of the protein that is NOT the polypeptide chain--these are atoms that form a chemical group held by a protein like a cytochrome (or a hemoglobin) Iron atom is in the center (can go between the oxidized and reduced state depending on whether or not it is carrying an electron) Each cytochrome has a slightly different heme to carry its electrons (thus thats why each one can have a different affinity for these electrons in this respiratory chain)

Cancer cells

Have a shorter cell cycle compared to non-cancerous cells....why? Maybe mitosis is faster? (NO) Perhaps G2 or S phase is shorter? -- can test this with the pulse chase experiment-- will see the second peak of mitotic cells much earlier than that in normal cell (but G2 and S phase are still the same length) Cancer cells have lost the ability to stop dividing Most other cells are able to transiently stop in G1--depending on the growth conditions and signals from other cells, they can continue into S phase, or they can exit the cell cycle and enter into G0 Cancer cells do not stop in G1

ETC and Proton Gradient

How do we get to ATP? As electrons are being passed to higher and higher affinity components, the energy that is released by that electron (going from loosely bound to tightly bound)-- this released energy is used to create a PROTON BATTERY-- each step can be harvested to move protons across the inner membrane, and thus build up a gradient of protons (also have a pH and charge gradient); protons from matrix end up in the intermembrane space Electron transport and proton pumping are "coupled"-- if protons cannot be pumped out, then electrons will not move down the ETC Ex.) If the proton gradient builds up too much, then not enough energy is released in moving electrons to allow for proton pumping, so both processes stop

DNA packaging

Human genomes consist of 6 billion base pairs Have to try and fit DNA which are three meters long into a nucleus that is 3 microns long --how do you get it to fit with out it become a twisted mess? The DNA wraps around specialized proteins called histones Nucleosomes = Particles composed of DNA wrapped around histone proteins (normally wrapped twice around 8 histone proteins) Chromatin = Consists of nucleosomes plus other associated proteins that help those nucleosomes form into more defined structures Can then be further compact into more complex structures, eventually to a chromosome (which is a contiguous stretch of chromatin) Histones are compose of a lot of amino acids that have basic side chains, which means under normal conditions, these proteins will have appositive charge on their R-group; thus, negatively charged DNA (due to phosphate atoms) will associate very tightly with these positively charged side groups on the histone proteins, which makes DNA spontaneously fold up on these histone proteins Can regulate how tightly the DNA will associate with the histone proteins just by changing charges

Heredity nonpolyposis colon cancer (HNPCC)

In Humans, is caused by mutations in the human homologs of MutH and MutL

Bacterial Cell Division

In eukaryotes, the cell cycle provides a way to separate the processes of DNA synthesis and mitosis and also gives the cells time to grow and prepare for division In bacteria, cell division and DNA replication are not always separated; this is feasible because bacteria don't have nuclei where the DNA is sequestered The chromosome is located mid-cell Then, the two replicated chromosomes start to pull apart when its time for the bacterial cell to divide Then, chromosomes pull apart to opposite side of the bacterial cell and a ring of FtsZ proteins form in the middle of the cell (sort of like cytokinesis)--which will constrict the membrane and cause the cell wall to infold....once two new cells are formed, fission is complete

Okazaki

Japanese researcher who discovered that the discontinuous DNA fragments will be synthesized one after the other on the lagging strand as the replication fork continues to open up and process...thus the fragments are called Okazaki fragments

DNA molecules

Just a contiguous double helix

G2 checkpoint

Just before cells enter mitosis Cells will only pass the checkpoint if the chromosomes have replicated successfully, if the DNA is undamaged, and activated MPF is present Enzyme that removes the inhibitory phosphate from the MPF is found at the G2 checkpoint and is what regulates whether or not the cells are ready for mitosis (so the enzyme will be inactive if the three conditions are not met with the cells)

The replication bubble

Just made up of two replication forks moving in two opposite directions DNA synthesis starts at an origin and proceeds in both directions Bacterial chromosomes have a single point of origin of replication At origin of replication, the DNA is unwound (begin to break hydrogen bonds between the bases to separate the two strands...) and replication will proceed in both directions Eukaryotic chromosomes have multiple points of origin of replication (in each origin, DNA begins to unwind, copy in both directions to both replication forks and eventually these replication bubbles will come together to finish the synthesis of the entire chromosome) Separating the DNA double helix, and copying each of the strands DNA is always synthesized in the 5' to 3' direction, so on one side, it is not big deal because that strand can be continuously replicated toward the replication fork...but the other strand has to move the opposite direction from the replication fork to be synthesized which will form gaps of single stranded DNA on one of the strands at the replication fork Strand copied continuously is the leading strand of DNA (also known as the 3' DNA strand) Strand copied discontinuously is known as the lagging strand of DNA (also known as the 5' DNA strand) Gap solution on the lagging strand -- will form fragments of DNA that are synthesized discontinuously (because still has to go 5' to 3' for synthesis)

Enzymes necessary for DNA synthesis in bacteria

Leading strand synthesis: 5' to 3' continuous replication towards the replication fork Helicase = the enzyme that unwinds the double helix DNA at the origin of replication; will bind to DNA and wrap around one of the strands of the DNA and start the slide along the the DNA double helix which causes the base pairs to be broken and the DNA to be unwound Right behind the unwinding are the single-strand DNA-binding proteins (SSBP) which are going to bind to the single-stranded DNA which is created by the unwinding and will prevent the base pairs from coming back together (stabilizes single strands) Then, RNA polymerase will synthesize the RNA primer...so RNA nucleotides are being added 5' to 3' using the template found in the single-stranded DNA...once RNA primer put down...then the DNA polymerase III can engage DNA polymerase III will work in 5' to 3' direction synthesizing the leading strand Sliding clamp= a "tether" that physically interacts with the DNA polymerase III and keeps it tethered to the template so it doesn't wander off to make replication very receptive and the polymerase will add nucleoside monophosphates very quickly to the end of the growing chain Topoisomerase = relieves the twisting forces by breaking a single phosphodiester bond in one of the strands of the DNA double helix while grabbing hold of the other strand and passing it through the little opening--helping to unwind all of the extra tension being formed in front of the replication fork DNA replication will stop almost immediately if there is too much tension in front of the replication fork Lagging Strand synthesis: Discontinuous synthesis from 5' to 3' of the Okazaki fragments away from the replication fork The primase will synthesize the RNA primer Still have DNA polymerase and the sliding clamp...but even though synthesis is occurring in 5' to 3' direction, actually happening in opposite direction of the way the replication fork is moving (due to the polarity of the nucleic acids) Lay down the Okazaki fragments...then open up the DNA helix up a little more until there is enough room from the primase to lay down another RNA primer...then we can synthesize the second Okazaki fragment...etc, etc DNA polymerase I will remove RNA (the RNA primer) and lay down DNA -- "repaving the road" (will be more frequently used in lagging strand synthesis since more primers...but still have to remove the primer(s) of the leading strand as well) Okazaki fragments not connected, so are bound together by the enzyme DNA ligase -- binding the last phosphodiester bonds between each fragment by the nucleoside monophosphates (intact DNA double helix) The entire complex of DNA synthesis is known as the replisome Looped out part shown in DNA synthesis as a whole is the lagging strand synthesis--because without the loop, polymerase would have to move in opposite directions (for the leading and lagging strands)...this way, they can both move 5' to 3' There are structural proteins that act as scaffolds for all of the enzymes...make sure they are working properly for DNA synthesis

Ionophores

Like valinomycin (lipid soluble) carries potassium ion, not protons across the membrane; stops ATP synthesis by decreasing the membrane potential So it is dissipating the charge gradient (rather than pH or proton) --can stop the store of energy in ATP just be adding positive charge into the membrane

What controls entry into mitosis?

Microinject cytoplasm from M-phase cell into one frog oocyte and cytoplasm from interphase cell into another frog oocyte The M-phase cytoplasm injected into cell...the oocyte immediately begins to progress into mitosis If interphase cytoplasm injected, nothing happens Conclusion: Must be something in the cytoplasm of the M-phase cells that promote mitosis...some molecule known as the mitosis promoting factor (MPF) The MPF is now known and is made up of two different proteins, making up a heterodimer -- cyclin and cyclin-dependent kinase (cdk) MPF needs to be active in order to send signal to cell that it is time to go into mitosis--becomes active by: having enough of the heterodimer (levels of cyclin fluctuate through cell cycle and get very high right before the cells are ready to enter into mitosis, but cdk remain constant through cell cycle); need to have the correct activating phosphate on one of the amino acid side chains on the Cdk and also needs to have the inactivating phosphate removed from the Cdk....then will enter into mitosis Will only go into mitosis when it is REALLY time--this will be regulated by various checkpoints

DNA synthesis

Most of the nucleotides in the cell are in the form of deoxynucleoside triphosphates and thus will be used in DNA replication They will be added to a template by an enzyme known as DNA polymerase (which catalyzes the reaction of adding the deoxynucleoside triphosphates to the DNA template)...in this reaction, pyrophosphate is released and our nucleoside monophosphates (A, G, C, or T) are what will be incorporated into our DNA double helix Almost all DNA polymerases require some sort of a primer in order to continue DNA synthesis...and the primer will either be RNA primer or DNA primer (has to be a nucleic acid) In this case, the DNA polymerase will use an RNA primer that is synthesized and is complimentary to the template strand and the DNA polymerase will extend the RNA primer in the 5' to 3' direction **ALWAYS synthesized (DNA and RNA) in the 5' to 3' direction -- so always adding to the 3' end (due to the attacking hydroxyl group to the incoming nucleoside triphosphate) How form RNA primer? -- DNA polymerases require the primer, but RNA polymerases do not...so we can use a particular RNA polymerase known as primase to synthesize an RNA primer complimentary to the DNA template so that we can kick-off DNA replication

Logic behind PCR

Multiple cycles of: Denaturation of DNA duplex (94 degrees celsius), then annealing of primers (55-60 degrees celsius) and last, DNA synthesis by polymerase (72 degrees celsius) One problem: most DNA polymerases denature at high temps This problem was solved with the discovery of a bacterium named Thermus aquaticus (found in hot springs), which makes a polymerase that is stable at high temperatures This stable polymerase is known as "Taq" polymerase and will synthesize the DNA at higher temps Cycle 1: Start with a solution containing template DNA that you want to amplify, synthesized primers, and an abundant supply of the four dNTPs and then denature the double-stranded DNA by heat (to have two separate single strands of DNA).....then lower the temp so that the primers will anneal (at cooler temperatures) to the template DNA by complementary base pairing...and then extension will occur by raising the temperature up again for "Taq" polymerase to function and add dNTPs to synthesize a complementary DNA strand, starting at the primer (which will happen in both directions and will make more product then you actually need Have to repeat the cycle of heating, etc to make the desired product After cycle 3 is when the desired product starts to appear...where you are amplifying exactly the DNA sequence that you want to amplify

Warburg Effect

Noticed that cancerous cells (tumor cells in body)--cells that are growing really fast/dividing rapidly, are not using respiration...using glycolysis--an aerobic fermentation Tumor cells go for the inefficient pathway PET tomography lets us see what cells in body are using glucose--will light up in high concentrations of glucose Bright spots= tumors (sucking up glucose) Using radioactive glucose to see where the tumors are..and stays in cell because using an analog of glucose, deoxyglucose...acts and looks like glucose but cannot be used in glycolysis Fooling the cell to take up deoxyglucose, so sucks it up like mad (will show us where the tumors are) These tumors are no longer cancerous when look at original areas after a certain amount of time because the analog used does not allow the tumor cells to go through glucose metabolism, thus not allowing the cells to rapidly divide (no longer malignant) In tissues that are dividing rapidly, whether or not there is oxygen, still going to go with the bail out pathway...settle for very little amount of ATP produced by each molecule of glucose; tumors reluctant at breaking up C--C high energy bonds in sugars...so doesn't want to do the reduction, but rather wants to build things using these bonds....using three carbon intermediate and shunting it into biosynthesis to have better substrate/ building blocks to generate new DNA, more protein, new membrane structure--bailout not just to oxidize NADH, but rather to keep all of the carbons together to generate better substrate for building new cells Anti-Cancer Therapeutic Drug --> need a transporter to suck up and to drag the pyruvate out of the cytosol into the mitochondrial matrix to produce CO2 so that the pyruvate cannot be used by the tumor cells/ new cells cannot be produced by biosynthesis

Metaphase

Occurs when chromosomes line up in the middle of the cell (complete the migration to the middle of the cell)

Cytokinesis

Occurs when cytoplasm is divided (already have divided the nuclei)-- actin-myosin ring causes the plasma membrane to begin pinching in until two daughter cells are formed

The effects of Activated MPF

Once the inhibitory phosphate is removed, the MPF will be able to go through with its enzymatic function and act as a kinase Phosphorylate chromosomal proteins (help with the final condensation to form those x-shape bodies)--initiate M phase Phosphorylate nuclear lamins-- initiate nuclear envelope breakdown Phosphorylate microtubule-associated proteins--activate mitotic spindle (and microtubules are able to attach to the sister chromatids at the kinetochores) Phosphorylate an enzyme that degrades mitotic cyclin--cyclin concentrations decline (which will turn off the cell's signal to "enter" mitosis, and it will be able to leave this stage in the cell cycle)

What makes the chromosomes move?

One clue: If treat cells with taxol, which prevents the depolymerization of microtubules, then mitosis stops Microtubules need to be dynamic and be able to polymerize in order for mitosis to continue Another clue: photo-bleaching experiment (FRAP)--looking at the movement of the microtubules; use fluorescent labels; watch these cells during anaphase that have a some tubulin subunits bleached (they are still there...we just can't see them) Results: The distance between photo-bleached area and the centrosome remained the same...but the distance between the chromosome and the photo-bleached area was reduced, thus...microtubules shorten only at one end, and that end is the kinetochore (chromosomes are being pulled toward the centromere) What would happen if it shortened at the centromere end instead? --ponder this Anaphase of mitosis current model: Microtubule depolymerization (the loss of alpha and beta tubulin subunits) pulls chromosomes at the kinetochores towards the poles-- tubulin subunits are pulled off from plus end to minus end by the ring of the kinetochore fibers....so as ring moves to the right, so will the chromosome (moving away from the centromere)

M-phase checkpoints

One determines if the chromosomes have attached to the spindle apparatus -- chromosomes that are not yet lined up on the metaphase plate would trigger this checkpoint until they line up on the metaphase plate, until there was tension between the microtubules of the mitotic spindle, and the sister chromatids are within the chromosomes Other point a little later in mitosis -- activated if the chromosomes don't properly segregate (if a chromosome stays behind on the metaphase plate)

Regulation between DNA and Histone Association

One way to promote chromatin opening is acetylation of histones (specifically adding the acetyl groups to the basic R-groups which normally carry the positive charge) Enzyme HATs = Histone acetyltransferases--will add acetyl groups to the basic R-groups and then the positive charge will be removed (go from positive to neutral charged proteins) -- association with DNA will not be as tight due to loss of attraction between opposite charges (beginning to decondense the chromatin) Enzyme HDACs = Histone deacetylase -- will remove the acetyl group from those side chains and return the chromatin back to it condensed form -- thus, the reverse reaction of HATs Only find histone proteins in eukaryotes (organisms with nuclei) Regulation of chromatin structure is crucial to what genes the cell expresses (what proteins the cell decides to make), and how an organism develops over time

Mitochondria

Organelles inside the cytoplasm of the cell--lots of them and come in different kinds of shapes Have two membranes that surround them: Outer membrane: Inner membrane: Has all the infoldings known as CRISTAE--which increases the surface area of the inner membrane (which thus allows there to be more ATP synthase--the inner membrane protein needed) In between the two membranes is known as the intermembrane space; the space inside the cristae is continuous with the intermembrane space Matrix: The other area within the inner membrane/ enclosed by the inner membrane (this is where pyruvate needs to go) Not always found as the compact little cigar shapes--can also be found, for example, wrapped around a flagella (such as is the case for sperm) Can be very diverse in appearance and more importantly they are PLASTIC--they are DYNAMIC, they change shape rapidly, grow, expand, contract, etc--all sorts of things depending on the cells needs Aerobic exercise can increase the number of mitochondria in muscle Cardiac muscle cell mitochondria have three times as many cristae as in liver cell mitochondria UK with "three-parent babies" -- use vitro fertilization technique using DNA from three people that could proven mitochondrial diseases; only inherit mom's mitochondrial DNA because the egg is what supplies the mitochondria to the embryo; if mom's mitochondrial genomes are mutated, we can transport mom's nucleus that has both mom and dad's genes to a donor egg with non-mutated mitochondria = a three parent embryo

Brown Fat

Our body's way to have adaptive thermogenesis- when we need to produce heat in our body; has special mitochondria (very abundant) that contain a special kind of proton pores known as uncoupling protein (UCP) in their inner membranes--another kind of "back door" approach Do not go to ATP synthesis due to UCP, but rather they dissipate that gradient and the energy stored in that gradient is released as heat Uncoupling protein sits as a pore on the inner membrane of the mitochondria Can convert white fat to brown fat

How to design primers for PCR

PCR primers (which will be DNA primers and not RNA primers) must be located on either side of the target sequence, on opposite strands DNA primers are more stable Found on the 5' end of each strand of DNA Need to read sequence of primer from 5' to 3' section Primers target template single stranded DNA, bind to it and allow DNA polymerase to take 3' hydroxyl of primer and extend it from the 5' end to the 3' end of the new strand to make complimentary bases

Watson and Crick

Postulated that due to the specific pairing of the nitrogenous bases, there is some possible copying mechanism for the genetic material Used bacteria because simpler than eukaryotes Could work like this: If hydrogen bonds between complementary base pairs are broken, the DNA helix can separate and each of these old strands of DNA can then serve as a template for a new strand that will be synthesized (and free nucleotides will attach according to complementary base pairing) These new strands will then polymerize (to form a sugar-phosphate backbone to restore the secondary structure of DNA) using the old strands as the template, and can have two double helices formed from this, each having one old strand of DNA and one new strand of DNA = semi-conservative DNA replication Others though may be other mechanisms of DNA replication -- Conservative replication = in which the bases flipped out, copies were made from the flipped out strands, and then the bases from the old strands flipped back in...making two double helices (one completely of old DNA strands and the other completely of new DNA strands) Dispersive Replication = strands will be cut up, somehow copied, and then reassembled in no particular order--all the new strands will be mismatched back together with the old strands

Interphase

Prior to mitosis The centrosomes will duplicate and the chromosomes will replicate into the two sister chromatids and the cell is then prepared to enter into mitosis

Chargaff's Rules

Prior to the 3-D structure of DNA known Say that ratio of adenine to thymine is approximately one--same percentage of each in a double-stranded molecule Say that ratio of cytosine to guanine is approximately one-- same percentage of each in a double-stranded molecule Thus, the ratio of pyrimidines to purines is approximately one Can be represented mathematically: A+G (purines) / C+T (pyrimidines) = 1 If the ratios are not equal (or not "1"), then it is probably single-stranded DNA or RNA

What is the genetic material?

Protein-- Think this because thought that the 20 amino acids (at least) would be enough diversity to offer the complexity needed to encode the vast number of traits in different organisms Others believed that nucleic acids in form of RNA and DNA encoded for this heredity material

Pyruvate Transportation

Pyruvate generated by glycolysis Pyruvate goes into this cycle in the presence of an electron acceptor, specifically oxygen (Glycolysis happens in the cytosol--liquid portion of the cytoplasm--thus, pyruvate is generated here...but has to get to the mitochondria!!) Transport pyruvate to where all enzymes are in the Krebs cycle-- Need to get out of cytosol and to the mitochondrial matrix for the Krebs cycle; needs to get through both the outer and inner membrane Pyruvate is cotransported into mitochondria with protons (to neutralize the charge) in order to continue with the metabolic cycle Can pyruvate cross through membrane? Small, but charged and hydrophilic...so not something membranes will be permeable to Outer membrane of mitochondria has a lot of pores...so can move up to anything smaller than 5,000 daltons in and out of the membrane no problem, including the pyruvate Inner membrane is an intact bilayer and therefore insoluble to ions, so we need transport proteins-- specific transport proteins bring pyruvate into mitochondrial matrix for next step of glucose metabolism

G0 phase

Quiescent state that cells can enter from G1 phase and stop cycling altogether

RNA structure

RNA hypothesis: that RNA came first (before DNA) Possibly came from Mars?? A ribose sugar (not deoxyribose sugar) with a 2' carbon having a hydroxyl group, thus making it highly reactive with other molecules and can even attack other parts of the RNA molecule and cause it to break apart, thus RNA is not a very stable molecule at all (compared to DNA) Has uracil as a base instead of thymine RNA normally doesn't form a double-helical structure, but it can have a significant secondary structure: RNA molecules form a stem-loop structure-- stem part is made up of the approximate shape of a double helix (base pairs forming hydrogen bonds...but the loops part has no hydrogen bonds); this RNA molecule is a part of a larger molecule known as tRNA (transfer RNA). which us used during cellar process of translation RNA molecules form a ribozyme structure-- big mix of both double helical structures and single strands of RNA that is all folded into one large complex secondary structure; this can carry out what we call "enzymatic" or chemical reactions--acting as an enzyme catalyzing the rearrangement of different bonds; different RNA molecules that act as this enzyme can do different things: Hammer-head ribozyme--engages in self-cleaveage to cut itself into multiple pieces Another RNA ribozyme that can act as an RNA polymerase--an RNA molecule that can serve to make copies of other RNA molecules (Able to carry out a lot of catalytic reactions, thus, scientists believed RNA came first and DNA came after)

FAD

Reduced to FADH2 by a hydride ion Can be observed in the lab because FAD is bright yellow (oxidized form) and FADH2 appears colorless (reduced form)

NAD+

Reduced with an H+ and two electrons to form NADH (an electron carrier) -- can also be reduced by a hydride ion (which is the same as what is written)

Sister chromatids

Replicated chromosomes after S-phase

DNA replication in eukaryotes

Same logic as bacterial DNA replication, but different protein names 2 different DNA polymerases (instead of same for both the leading and lagging strand) Unique problem: What happens when the replication fork reaches the end of the linear chromosome? (Because bacterial chromosomes are circular and thus the two replication forks will merge and with topoisomerase, the circular chromosome will be completely replicated no problem) RNA primer will be removed, which then leaves a space at the end of the lagging strand template where there is no 3' hydroxyl for a DNA polymerase to extend from (so no DNA synthesis can occur once this RNA primer is removed --chromosome is shortened over time How do we solve the problem of the telomeres (the ends of the chromosomes) not being replicated? Many cells use telomerase to solve the end replication problem-- The ends of linear chromosomes (telomeres) have many copies of a repeated sequence (in humans it is TTAGGG) This repeated sequence is helpful because telomerase is composed of both an RNA and DNA component...the RNA component is a template that is complimentary to the DNA nucleoside at the end of the chromosomes, so telomerase will bind to the end of the lagging strands and the RNA is complimentary to the end of those chromosomes and since it is a enzyme, it can catalyze the addition of DNA nucleoside monophosphates to the end of the chromosome Telomerase synthesizes a little bit of DNA, moves over, and then synthesizes a little bit more of DNA...and end up at the end with one very long strand of DNA at the end of the lagging strand--what you have done is bought yourself some more room for additional RNA primers to be synthesized in the next cell cycle and for the DNA polymerase to be able to extend that Adding more DNA to fix what was taken away due to the end replication problem

The Human Genome Project

Sanger sequencing was used to sequence the genome

Nucleic acid polymerization

Seems like a simple condensation reaction where two nucleic acids come together and form water...this would only be true of nucleotides existed in cells as nucleoside monophosphates....but they actually exist as nucleoside triphosphates Have three phosphates attached to the ribose and the base--and the bonds between the phosphate groups are extremely high in energy (potential energy)--these are phosphodiester linkages (a phosphate with two ester linkages) Cell uses the energy within these monomers to drive the process of nucleic acid polymerization forward and prevents it in engaging in the reverse reaction Add to the 3' end of the chain--the hydroxyl group will attack the nucleoside triphosphate; this reaction is catalyzed by the enzyme DNA polymerase, and what happens is that pyrophosphate is produced and then a nucleoside monophosphate is added to the growing chain The pyrophosphate then reacts with an enzyme known as pyrophosphatase which will produce two inorganic phosphates plus a ton of energy-- this energy is what makes sure the polymerization reaction in the cell almost irreversible (only occurs in the forward direction) -- once you add a nucleoside monophosphate to the growing chain, you're not going to remove it unless something else intervenes Coupling high energy bonds within the nucleoside triphosphates and the breaking of one of those bonds to drive the polymerization reaction forward

The Sanger Method

Similar to PCR but two very important differences: 1.) DNA to be sequenced is mixed with a single primer(not trying to make a billion copies of the DNA like in PCR, but rather just trying to figure out what the actual sequence of the bases are in the DNA sequence), DNA polymerase and dNTPs 2.) Use special chain-terminating dideoxynuceloside triphosphates (ddNTPs) in the reaction at low concentrations-- in a ddNTP, the 3' hydroxyl will be missing and is replaced with a hydrogen (so no DNA synthesis can occur after this base) -- When ddNTPs are incorporated into DNA polymer, they prevent the formation of a phosphodiester linkage with an incoming nucleotide Modern Sanger sequencing: Use normal dNTPs and fluorescently labeled ddNTPs in polymerization reactions Have an excess of dNTPs and a small number of ddNTPs and each base will be a different color with different fluorescent labels Incorporation of a ddNTP stops the polymerization reaction -- Use A, G, C or T and see where the polymerization stops and collect the DNA strands that are produced (and will have different sized products)...and then separate them in capillary tube gel electrophoresis At the end of the capillary tube, there is a fluorescent detector that can detect the identity of the ddNTPs and thus the sequence of the synthesized DNA is determined The shorter fragments will pass faster through the agarose in the capillary tube (and be found closer to the 5' end of the DNA molecule) Smallest DNA fragments represent the ones where the ddNTPs where incorporated very early on in the synthesis reactions (closer to the 5' end of the DNA molecule) Read the chromatogram backwards to find the 5' to 3' sequence of the non-template DNA Primer is not included in the non-template DNA and thus will not be shown in the chromatogram

Anaphase

Sister chromatids separate and the chromosomes will be pulled to opposite poles of the cell

Other gradients

Some of the protons are critical in order to get most of the substrates across the membrane, because most of them are charged (such as getting ADP in and ATP out) ADP in, ATP out is driven by voltage difference across membrane because ADP carries less negative charge than ATP

Figure out how the ETC works

Start with NADH and move it down the chain to oxygen...we are trying to reduce the oxidized oxygen to H20 Electrons flowing into this system from those reduced carriers that generated degradable sugar Electrons flow out of the system by being transferred to oxygen Use a poison-- if it blocks at a certain point in the chain, it prevents the passage of the electron from the prior step to the next step in the ETC--thus, the carriers prior to this poison cannot give their electrons to the next carrier and thus remain reduced (thus, will end up building up NADH or FADH2 and will not be able to reoxidize it to NAD+ unless we shunt glucose for fermentation) Downstream from the poison, the carrier that has the electron will release it to the next carrier and become oxidized, and so on-- eventually pass to oxygen, but they will have to remain oxidized because we have blocked their reduction (and thus stop using oxygen because we won't have anymore electrons to pass to the oxygen to reduce it to water) Using inhibitors such as a poison and determining which carriers are oxidized or reduced, we are able to see that it is possible to reconstruct the order of the electron carriers

Electron Transport Chain

The electron carriers will transfer their electrons through a stepwise chain of compounds with successively higher affinities for electrons -- at each step in the transfer, some energy will be given up Take NADH and FADH2 to feed the electrons to the new carriers (new centers for oxidation and reduction) These new carriers are integral membrane proteins located on the inner membrane of the mitochondrion (two exceptions: Q and Cytochrome C) Form a chain such that each carrier in that chain has an increasingly higher affinity in for the electron, thus energy is released in the passage

Prometaphase

The nuclear envelope begins to break down The spindle fibers originating from the centrosomes will begin to connect to the chromosomes at a particular protein DNA structure known as the kinetochore -- these microtubules will push and pull the chromosomes around the cell until they line up in the very middle of the cell known as the metaphase plate

Telophase

The nuclear envelope re-forms, and the mitotic spindle apparatus disintegrates

Cytochrome C

The other exception that is not an integral membrane protein (as well as Q) Soluble small protein that travels along the surface of the inner membrane in the mitochondria on the intermembrane face Can detect if it is reduced or oxidized by the differences in the absorption spectrum (of light)--will absorb more light when in reduced state (and thus when carrying an electron)??

Mitosis

The replicated chromosomes, at the beginning, condense to form the x-shape structures that are held together by a centromere in the center (also the point where we are going to assemble the kinetochore...and the kinetochore is where the mitotic spindle is going to attach and start to provide tension to pull the sister chromatids apart) During mitosis, sister chromatids separate and two daughter cells are formed by cytokinesis Important: You end with the SAME number of chromosomes (in each cell) that your started with...so if you started with 4, you'll finish with 4 Each daughter cell will have the same exact genetic information

Carriers in the ETC

They form complexes in the inner membrane of the mitochondria Oxygen is the final acceptor in the ETC and has the highest affinity for electrons (this is why aerobic respiration depends on oxygen)

G1 checkpoint

This is where cells make the decisions as to if they will continue in the cell cycle, replicate their DNA and divide into two new daughter cells OR enter into the quiescent state known as G0 The cells pass the checkpoint if: The cell size is adequate, the cells have sufficient nutrients, social signals are present, and the DNA is undamaged Signals arrive in the form of growth factors coming from other cells or the cell itself deciding if it should go on and divide If these signals are present, going to cause two factors to increase: the G1 cyclin and the protein known as E2F G1 cyclin will bind to G1 cdk to form a heterodimer and the cdk will be phosphorylated with both activating and inactivating phosphate Cyclin/cdk complex's target is the Rb which E2F is bound to Rb (retinal blastoma protein) is a molecular brake (keeps the E2F from doing its job--act as a transcription factor and to promote the expression of genes that are important for S-phase When appropriate signals are present, the inactivating phosphate is going to be removed from cdk and then active cdk will be able to phosphorylate Rb, which changes Rb's shape so that it then releases E2F E2F is now free and will trigger the production of S-phase proteins (because it is a transcription factor--which binds to DNA double helices and promote expression of genes that are important)

Cellular Respiration taken from Pyruvate

Two paths that pyruvate can go through: Aerobic: Presence of oxygen (abundant oxygen available in the cell) -- goes through Krebs cycle and electron transport chain and oxidative phosphorylation to make a ton of ATP-- highly efficient process Anaerobic: No oxygen; will not begin Krebs cycle; instead shunted into relatively inefficient pathway--little ATP comes out of this pathway

Proton Battery

Used to power a molecule motor which is known as ATP synthase (an enzyme that takes ADP and adds a phosphate group, so it is storing energy in the form of ATP) --enzyme is simply a molecule motor powered by the proton battery ATP synthase is a molecular machine that works like a turbine to convert the energy stored in a proton gradient into chemical energy stored in the bond energy of ATP The protons move down their electrochemical gradient drives a rotor that lies in the membrane....has an enter channel to bind to the rotor which then spins it...once it has made a full rotation, it then reaches the exit channel to the other side of the membrane....the energy stored in the proton gradient is converted into mechanical/ rotational energy The rotational energy is transmitted via a shaft attached to the rotor that penetrates deep into the center of the F1 ATPase-- which catalyzes the formation of ATP F1 ATPase portion of ATP synthase has been crystallized Can work in either direction (meaning it can hydrolyze ATP to pump protons back across the membrane in high concentration of ATP and low proton concentration) The protons in the gradient enter into the point of the ATP synthase known as the F0 unit--proton will enter into channel and binds to site in channel which forces ring of protein subunits to turn one notch-- the stator is stable in the membrane, but the F0 spins and is connected to the rotor mechanism, which serves as a paddle that goes "whack whack" and is being turned around by top portion (F0 unit)--as rotor spins, it "whacks" the stator at each perceptive location of F0 unit, so when it gets whacked it changes conformation of the F1 unit (from the mechanical energy) which allows the binding of ADP and phosphate, then the catalysis creating the bond and finally the release of ATP into the matrix

Mitochondria Origin

Why have two membranes/ two bilayers? Endosymbiont hypothesis: Some ancestral cell very early in eukaryotic lineage had developed a nucleus but then took up some bacterium by phagocytosis, and then that bacterium become vaguely associated with that cell...and then would pass on to future generations of those eukaryotic cells-- gives idea where those two membrane could've come from--if cell is engulfing that bacterium, then outer membrane of mitochondrion would be the cell plasma membrane and inner membrane of that mitochondrion would be the bacterial plasma membrane Phagal association of that bacteria inside the cell plasma membrane thats been budded off by the phagosome, as a way of eating things on the outside of the cell Mitochondria has its own DNA-- usually circular, which is a hallmark of many bacterial genomes DNA critical for growing new mitochondria or passing mitochondria on during cell division to the daughter cells DNA codes for translation of proteins and enzymes necessary for mitochondrial respiration

Can you synthesize DNA in a test tube?

Yes! And do not need everything necessary for bacteria/ naturally Need: Primase to make primers, DNA polymerase, dNTPs, and DNA template Do not need: Helicase, topoisomerase, SSB proteins, DNA ligase, telemorase or clamp Simplified way of DNA synthesis in a test tube was figured out by Kary Mullis in 1983 Process he came up with is called polymerase chain reaction (PCR) -- Used to make millions of copies of a short DNA sequence from 50 base pairs to >5 kilobases and you can make these copies even if your DNA sequence is part of a complex mixture (like the human genome) One catch: You have to know the sequence of the DNA that you're trying to amplify-- because PCR circumvents the need for the enzyme primase...instead of putting primase in the reaction, you simply synthesize primers in a laboratory (DNA primers)...and these primers with the DNA template, DNA polymerase and dNTPs will serve as the primers necessary for DNA polymerase to carry out DNA polymerization Realized can do this without DNA helicases if heating up the DNA to just the right temperature that will disrupt the hydrogen bonds between the base pairs