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Biology outcomes for 2nd midterm.pdf

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Biology 1002B
Tom Haffie

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Lecture 10 - Evolution of Eukaryotes Meaning of endosymbiosis • A living organism living within the cell of another living organism - they help each other out. Origin of endomembrane system, nuclear membrane, ER, etc • The endomembrane system (nuclear envelope, ER) was thought to have been derived from infolding of the plasma membrane (supported by the face that ER is connected to the nuclear envelope. • Having a nuclear envelope and a nucleus allowed for compartmentalization and was critical • **Development of the n.e and ER is NOT part of endosymbiosis. Origin of mitochondria and chloroplasts • Mitochondria and chloroplasts are not derived from the same place the nuclear Bacteria that can envelope was derived undergo oxidative • Mitochondria is said to have derived from an aerobic bacterium after a larger phosphorylation primitive cell brought it in through phagocytosis - it being the mitochondria. • Chloroplast is said to have descended from a cyanobacteria May have been an anaerobic bacterium that • ‘Aerobic bacterium’ that had mitochondria engulfed a cyanobacteria (lead to plants and such) had an endomembrane system Top branch gets you plants, algae, etc • •Bottom branch gets you animals, fungi, etc • So, aerobic bacterium (free living bacteria) was engulfed and then evolved into the mitochondria over time • A subset of those primitive cells that engulfed aerobic bacterium then engulfed cyannobacteria Evidence supporting theory of endosymbiosis 1) Morphology - mitochondria look like bacteria (similar shape and what not) AND chloroplast kind of looks like cyannobacteria 2) Mitochondria/chlorplast divide within a living cell identical to the way bacteria divides 3) Mitochondria and chloroplasts are the only organelles within eukaryotic cells that have electron transport chains which means they probably did electron transport when they were free living cells as well 4) They have their own genomes (circular chromosomes) 5) They have transcription and translation machinery - have their own ribosomes and make their own proteins. 6) Mitochondria and chloroplast structure is very similar to prokaryotic cell structure Factors driving development of early eukaryotic cells • OXYGEN - about 2.2 bya cyanobacteria developed and they could do oxidative phosphorylation (split water - release oxygen into atmosphere) • This paved the way for aerobic respiration --> provides MUCH more ATP than glycolysis and fermentation --> cell can now make more energy. Why eukaryotic cells can be larger and more complex than prokaryotes • Bacteria and Archaea have their centers of oxidative phosphorylation on their plasma membranes. So, if they try to get bigger, they need more centers of oxidative phosphorylation to give them more energy. • Problem is that prokaryotes’ plasma membrane’s surface area increases as a function of radius SQUARED whereas the volume increases as a function of radius CUBED A = 4 πr 2 • Volume gets bigger much quicker than surface area of plasma membrane and so the cell tries to compensate by putting more centers of oxidative phosphorylation on the plasma membrane BUT eventually it runs out of space 3 V = 4/3 πr on the p.m and that is what limits the size of prokaryotes (not being able to produce enough energy to support a larger cell) • Prokaryotic Cells have a HIGH plasma membrane area to volume ratio • However, eukaryotes can be much bigger because you have mitochondria and each mitochondria has many ox-phos centers and so you can produce MUCH more ATP and support a larger cell/genome LOW plasma membrane surface area to volume in eukaryotes. • • Eukaryotic cell has more energy to invest into protein synthesis and as a result can generate/express more proteins that are often more complex. Evidence for lateral gene transfer from organelles to the nucleus Genes that code for proteins found in mito/chloro are not found and expressed • in the nucleus ( e.g - proteins that make up complex 1) General idea about how lateral gene transfer is detected (Southern Blot) • Isolate genomic DNA , run it on gel (make DNA single stranded) Add a single stranded DNA probe and see if hybridization occurs • • You can then see what genes you have in that genome. • So you can isolate mitochondrial DNA, and nuclear DNA and add the probe of Oxidase 3 (mitochondrial gene) If the gene is found mtDNA but not the nDNA - Lateral gene transfer has not • occurred • If found in nDNA and not mtDNA - lateral gene transfer has occurred • If found in both, LGT occurred and both genomes have a copy of this gene. Hypothesis for why genes move to the nucleus from organelles (lateral gene transfer) • Coordinated control / put them under tighter nuclear control • Integrates the metabolism of the entire cell (see lecture 11) Possible reasons why certain genes have NOT moved to the nucleus from organelles • Lecture 11 Role of cpn60 in tracing endosymbiotic and lateral gene transfer event in eukaryotes • cpn60 is a mitochondrial gene found in the nucleus (LGT) • Giardia (creature with no mitochondria) has cpn60 in its nuclear genome so at one point it did have mitochondria but now cpn60 has no function in giardia. • This means that the ancestor that gave rise to giardia and other eukaryotes had mitochondria with the cpn60 gene but giardia got rid of it to better adapt to its life style. Lecture 11 - Intro to Prokaryotic Gene Structure Relative sizes of typical mitochondrial, chloroplast, and nuclear genomes Nuclear genomes(linear) > Chloroplasts Genome(circular) > Mitochondrial Genome(circular) • If genome is 5000kb, and contains a total of 5000 genes, then every gene is made of 1000 base pairs (5,000,000/5000) and every amino acid is 3bp, so amount of amino acids in a gene of this organism is 333bp (1000/3) Possible reasons why modern organelle genomes have become dramatically smaller over evolutionary time • Got rid of genes that were useful for free living bacteria (genes coding for flagella) • Got rid of redundant genes that were also in the nucleus - genes involved in glycolysis • Certain genes have been deleted • Hosts in which their mitochondria has suffered a mutation / deletion such that it has lost a bunch of genes, those genomes will be easier to replicate and those organisms will have a selective advantage • Thus, it is in the organisms favor to get rid of all the genes they can in the mitochondria so long as they are redundant -- one way to get rid of these genes is by mutation and deletion, another way is to send them to the nucleus through Lateral Gene Transfer. Possible reasons why genes have moved to the nucleus from organelles over evolutionary time • Coordinated control • Organelles, mitochondria and chloroplast are involved in electron transport and oxygen metabolism that generates reactive oxygen species. ROS’s are very Oxygen + electron = reactive and mutagenic and damage DNA and so it makes sense to get your Reactive oxygen species DNA out of the organelles and into the nucleus -- get DNA away from ROS’s • To avoid any kind of cellular rejection system • However, main reason for LGT is sexual recombination - getting organelles out of the chloroplast and into the nucleus allows them to participate in sexual recombination and generate diversity. Possible reasons why certain genes have NOT moved to the nucleus from organelles vs. eukaryotic nuclear environments • They need to have local control • Transporting proteins out of the cytosol into organelle may be to much of a hassle (too hard to move the protein - maybe too big -- size) • Structure of genes in the organelle may not work in the nucleus - this is because the general environment in our organelles is essentially prokaryotic, whereas the environment in the nucleus is a eukaryotic environment - so it is a big change for the gene to go from one to the other. Could be due to chance - genetic drift. There hasn’t been enough time yet for all • of them to move - evolution is STILL occurring. Maybe in a million years there will be less DNA in mitochondria. Rubisco structure and assembly from components coded by different genomes Complex protein components ( proteins involved in Electron transport/Calvin • Cycle [Rubisco] ) are coded by DIFFERENT genomes. • Some parts of the protein will be coded by genes in the nucleus, whereas others are coded from genes in the chloroplast. Basic structure and function of RNA polymerase and ribosome • Ribosomes are primarily RNA machines - it is the RNA that is catalytic ▯ The protein is structural • In prokaryotes, RNA polymerase understands the information of the promoter - as a result, it binds to it and begins to transcribe ▯ RNA polymerase reads 3’ to 5’ and synthesizes RNA 5’ to 3’ ▯ - initial end of the RNA transcript is 5’ and the other end is 3’ Examples of complimentary base pairing in gene expression • Once mRNA is formed from transcription, it base pairs with itself - folding is critical for its structure. • tRNA complimentary base pairs with itself to obtain proper 3D shape AND it also pairs with mRNA (codon with anticodon) • rRNA in ribosome complimentary base pairs with itself to achieve proper 3D structure Note: Mitochondria and chlorplast have their own gene expression machinery - they • both have ribosomes • Stop codons stop TRANSLATION , not transcription Lecture 12 - Prokaryotic Gene Function (ISO) Identify the sequence of standard “start” and “stop” codons • Start codon : AUG Stop codons : UAA, UAG, & UGA • Identify the function of “start” or “stop” codons • AUG codes for the amino acid methionine and is the first codon translated in ANY mRNA in both prokaryotic and eukaryotic cells. • Stop codons (do not code for amino acids) act as “periods” indicating the the end of a polypeptide-encoding sentence. When a ribosome reaches one of the stop codons, polypeptide synthesis stops and the new polypeptide chain is released from the ribosome. Compare the overall gene expression of prokaryotic vs eukaryotic cells Eukaryotic Gene Similarities Prokaryotic Gene Expression Expression • No SD box - instead, • Start codon is the • SD box helps initiate small subunit of same in translation translation ribosome recognizes • TATA boxes • Termination sequence 5‘cap, binds to it and Same termination is transcribed - forms • moves along the ‘signal’ in translation. loop and ends mRNA (scanning) transcription • Cleaving of • no micro RNA genes. polyadenylation signal signified termination of transcription Lecture 12 - Prokaryotic Gene Function Relative location of such DNA sequence “signals” as promoter, 5’ and 3’ UTR, “SD BOX”, start codon, stop codon, transcription terminator etc.(PROKARYOTIC) Gene begins with area called the promoter • •In between the promoter is the TATAbox (TATA box is upstream of the In a DNA strand where transcription start point -- transcription start point is essentially where the promoter is on the left promoter ends) side, upstream is to the •The Transcription unit goes from the transcription start point to the transcription left of, and downstream is stop point to the right of. •The Gene includes the promoter AND the transcription stop point. Note: Transcription •5’UTR - untranslated region - stretch of DNA following the Promoter that is transcribed into mRNA but is NOT translated (downstream of the promoter, starts here, not at the upstream of the start codon) start codon. •Down stream of the 5‘UTR is the start codon , at the end of the gene is the stop codon. •Downstream of the stop codon is the 3‘UTR - transcribed - not translated •At the end is the terminator - specific DNA sequence for a gene that signals Acts after it is transcribed the end of transcription of a gene. •SD box is upstream of the start codon (found within the 5‘UTR) Region of DNA that once transcribed into Mechanism by which each signal is interpreted, or understood by cell mRNA, base pairs with (prokaryotes) ribosomal RNA to help Promoter the initiation of translation •Sequence of promoters can be very attractive and so polymerase binds very stably or promoters can have sequences that less attractive and thus less efficient at transcription. • Promoter = signal , Interpreted by: RNA polymerase Terminator •In prokaryotes, terminator sequence in the DNA gets transcribed and ends up in the mRNA - this mRNA then complimentary base pairs with itself to form a loop (a hairpin) structure and the loop is what signals RNA polymerase to stop. • Loop leads to a signal that causes mRNA to dissociate away from the DNA template - loop destabilizes mRNA thats bound to the DNA and so mRNA falls off and termination of transcription occurs • SIGNAL IS IN DNA (Terminator DNA sequence) , BUT IS UNDERSTOOD AS RNA (RNA polymerase understands the ‘loop’ means stop) SD Box • SD box is found in DNA inside the 5‘UTR, once transcribed, its RNA base pairs try to bond, however tRNA will not bond with them because translation starts at the start codon (downstream of SD box) and ribosome moves right (away from SD box) • However, rRNA in the small subunit of the ribosome will pair with the mRNA to help initiate translation - SD box found in DNA but is understood/significant as RNA. • The release factor is ALWAYS trying to get into the “A” site of the ribosome but is always outcompeted by tRNA. However, no tRNA binds to the stop codon and so the release factor has time to get in and BIND (It does not base pair - it is a protein) -- as a result of the binding of the release factor, translation stops. Relationship between DNA sequence of signals and their function (how would low efficiency promoters be different than high efficiency promoters?) Promoters • Transcription starts at the startpoint (0) , and negative sequences indicate where the sequences are relative to the startpoint • At the -10 sequence (upstream of startpoint) their is a lot of AT sequences - this is where the bubble begins (bubble is formed when Promotor attracts RNA polymerase and RNA polymerase interacts with the DNA) • -35 sequence is the beginning of the promoter (upstream of -10 sequence) • Some promoters are very attractive - their sequence is such that polymerase makes a very stable bind and initiates transcription very frequently. • Other promoters have different sequences and are less attractive and thus less efficient at transcription • Promoters have a general common sequence and structure but are also quite variable and can drive transcription at different rates. Terminator • If one terminator stops transcription 60% of the time and another only stops 40% of the time - this could be due to the length of the terminator sequence, longer = more stable and therefore higher termination rate • One terminator sequence can have more G’s and C’s (3 hydrogen bonds b/w them) and so the loop will be more stable and as a result you have a more efficient terminator. Characteristics of promoters that require a particular position and direction • If the promoter of gene b is on the right, then polymerase will bind to to the right side of the blue gene and read 3’ - 5’ on the top strand • If the promoter is on the left side of blue gene (gene b) then bottom strand will be the template strand that is read 3’ to 5’ • **The strand that becomes the template strand depends on the position of the promoter. Base sequence of start and stop codons as mRNA and DNA • Start codon in DNA : 3’ ”TAC” 5’ , in mRNA : 5’ “AUG” 3’ • Stop codon in DNA: 3’ “ATT” 5‘ , 3‘ ATC 5‘ 3‘ ACT 5’ in mRNA : 5’ “UAA” 3‘ 5‘ UAG 3‘ 5’ UGA 3’ The location of various signals given a diagram of gene expression | 5‘UTR | (Promoter) (SD Box)(Start Codon) (Stop Codon) (Terminator) Note: • Start codons are in DNA but are understood as RNA • Start codons are NOT the start of transcription, they are the start of translation • Start codon is NOT the first three bases transcribed • Terminator is downstream of stop codon (terminate AFTER your transcribe stop codon) • +1 nucleotide is the first nucleotide transcribed (right after promoter) • 5‘UTR - transcribed but not translated - upstream/to the left of of start codon • Some amino acids have MULTIPLE codons that code for them. Mechanism by which each signal is interpreted, or understood by cell (prokaryotes) - continued • Start codon (mRNA) attracts the first initiator tRNA (that codes for methianine) into the P-site of the ribosome and that initiates the process of translation •To the left of the start codon is the UTR (mRNA to the left of start codon is UTR) •These bases want to base pair, so in BACTERIA (prokaryotic) the ribosomal RNA in the small subunit of the ribosome pairs with those base pairs (in the blue circle) - this sequence is called the SD box • SD Box - region on DNA that once transcribed into mRNA, complimentary base pairs with ribosomal RNA to help the initiation of translation • SD box is found in DNA but not ‘understood’ as DNA • Ribosome moves from the 5’ to the 3’ end of the mRNA and Ribosome eventually reaches the stop codon - no tRNA for stop codon, so • release factor(a protein) has a chance to get in and BIND in the A-site (it does not base pair) - translation stops. (stop codon in DNA, understood as RNA) Prokaryotic cell VS Eukaryotic cell In eukaryotes - transcription and translation are separated by the nuclear • membrane which is NOT present in prokaryotes • In prokaryotes, translation begins as soon as there is any mRNA available - as soon as the SD box is transcribed, then ribosomes can jump on there and begin translating Polymerase is making the message and at the same time, ribosome is • translating this message. • Multiple polymerases can be transcribing the SAME gene at the same time, as fast as they can, as fast as the promoter will allow. • Multiple ribosomes translating every message all at the same time. • Ribosomes are slower/bigger in eukaryotes and use method of scanning. (no SD box) In this picture Polymerase moves from right to left (3’ to 5’) creating mRNA where the 5’ end is furthest away from the DNA (blue line) Ribosome translates from 5’ to 3’, therefore ribosome closest to the strand has been translating the longest and will have the longest polypeptide chain coming out of it. Lecture 13 - Prokaryotic Gene Regulation (ISO) Identify the main features of bacterial operons • Operon - cluster of prokaryotic genes and the DNA sequences involved in their regulation Operator - short segment that is a binding sequence for a regulator protein • (repressor) When a repressor (regulatory protein) binds to the DNA, the likelihood that • genes will be transcribed is GREATLY reduced. • Other operons are controlled by regulatory proteins called activators - when bound to DNA, they increase likelihood of transcription. • Each operon is transcribed as a unit from the promoter into a single mRNA - so the mRNA contains codes for several proteins • A cluster of genes transcribed into a SINGLE mRNA is called a transcription unit Identify the function of repressor proteins • Binds to to the operator and as a result RNA polymerase is blocked from binding to the promoter (lac repressor acts as a road block) Identify location of various components of the lac operon Lecture 13 - Prokaryotic Gene Regulation DNA signals in RNA-coding genes •RNA-coding genes are NOT translated - so the only DNA signals they need are a promoter and a terminator. •No stop codon in RNA-coding genes - basically have no signals that would be understood during translation. •tRNAS code for the anticodon that is used during translation. DNA sequence of anticodon in tRNA gene, given the codon •Tryptophan (Trp) gene (top drawing) has only a promoter and a terminator UGG •On the DNA strand (3’ -> 5’) , RNA polymerase transcribed the gene and makes tRNA (5’ -> 3’) - CCA •Base pairing MUST BE complimentary and anti-parallel so when tRNA pairs with mRNA (5’ -> 3’) , the tRNA must be 3’ -> 5’ (ACC) •Off the template strand of DNA read 3’ -> 5’ , the DNA base pairs of the anti codon were GGT if tRNA is 5’ CCA 3‘ (before folding) Then DNA was 3’ GGT 5‘ • Trp mRNA codon is 5’ UGG 3‘ So tRNA is 3’ ACC 5‘ (in folded form - when pairing with mRNA) Likely effect of base sequence substitutions in various DNA signals •If you mutate the promoter you could inhibit polymerase binding or decrease the efficiency of the promoter OR the mutation could increase the efficiency of the promoter and make it more attractive to polymerase. •If you mutate SD box you could make the SD box more functional - more attractive to rRNA or less functional - less attractive to rRNA Mutation to the start codon just screws it up - you cannot make the start codon • ‘work better’ or more efficient (it is a lethal mutation) •If you mutate the stop codon into another stop codon, nothing happens, BUT if Stop codon is you turn it into a different amino acid, then translation won’t know to stop at that point. The ribosomal machinery will continue to read through and it won’t stop understood by TRANSLATION until it finds the next in frame stop codon. As a result, over •If mutation occurs in the terminator, the effect will depend on whether the loop evolutionary time, many genes have got 2 or 3 (hair pin) is strengthened and as a result the terminator becomes more efficient. OR the mutation could destabilize the loop - terminator becomes less efficient. stop codons in frame at their ends. Change in amino acid coded, given a change in the DNA sequence (and Genetic Code table) Silent mutation - base-pair substation mutation that destroys one codon but • creates another codon that codes for the SAME amino acid (no effect) •Missense mutation - base pair substitution results in a different amino acid being coded - nothing serious happens if new a.a is similar enough to the old one, but for e.g if an amino acid that is hydrophobic is replaced with one that is hydrophilic - this could seriously affect protein function. (more severe than Or a negatively silent) charged A.A for a positively charged one. •Nonsense mutation - base-pair substitution results in the formation of a stop codon - this could result in a protein that will PROBABLY be too short and will PROBABLY be non-functional (more server than above two) In/Del mutations - addition or loss of a base pair. If a base pair is added, the • entire reading frame shifts right (shifts downstream) and so a whole new set of amino acids are made (very severe) Note Start codon determines which three amino acids end up being paired together - • start codon sets the frame. • There can be three possible reading frames on a strand of DNA , e.g : 1)CAAATG ACC.. --> 2)AAA TGA CC --> 3)AAT GAC --> 4)ATG ACC ▯ In 4) your are back to the original reading frame Could possibly occur Therefore there are 3 possible reading frames on a strand of DNA as a result of three • additions or three deletions The location of various signals given a diagram of gene expression (Other codons for other a.a’s) (Promoter) (SD Box)(Start Codon) (Stop Codon) (Terminator) •Promoters are NOT transcribed // SD box IS transcribed /NOT translated •Start codon IS transcribed and translated •Stop codon is transcribed but NOT translated •Terminator is transcribed but NOT translated Basic structure of lac operon •Going from upstream to down stream: • LacI - regulatory gene, codes for Lac repressor protein • Promoter - Where RNA polymerase binds • Operator - Where Lac repressor binds • Lac Z - encodes the enzyme B-galactosidase Lac Y - encodes the enzyme Permease • • Lac A - encodes the enzyme Transacetylase Mechanism of action of lac repressor • Lac repressor is encoded by the regulatory gene : lacI • LacI is upstream of the promoter/operator • When there is no lactose in the medium , the lac repressor binds to the operator and prevents RNA polymerase from binding to the promoter Function of lac operon in the presence, and absence, of lactose Absence: • lacI is transcribed and translated forming an active lac repressor (a protein) • Lac repressor binds to operator and blocks transcription • Transcription of structural genes occurs rarely (repressor occasionally falls of of operator temporarily) and so a few molecules of each enzyme is being made Presence: • Beta-galactosidase molecules (already present in the cell) convert some lactose into the inducer - allolactose (isomer of lactose) • Allolactose binds to the repressor, inactivating it by altering its shape so that it cannot bind to the operator • RNA polymerase binds to the promoter • Transcription of lac operon (Z,Y,A) structural genes occurs • Translation produces the three lactose catabolism enzymes purple hexagon - lactose --> Pink - allolactose Note • Our mitochondria have some operons (otherwise operons are only in prokaryotes) The lac operon is an inducible operon - its normal state is off • • lac I is independent of Z,Y,A and has its own promoter/terminator..etc • Lac I repressor protein is a dimer • Lac repressor creates a loop of DNA that prevents polymerase from transcribing the operon Possible location of mutations in lac operon Phenotype that would arise from a given mutation in lac operon under given conditions Lecture 14 - Eukaryotic Genes Basic structure of eukaryotic vs prokaryotic cell w
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