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Biology 1002B - Term Test _2.docx

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Department
Psychology
Course
Psychology 1000
Professor
Dr.Mike
Semester
Winter

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BIO 1002B TERM TEST 2 LECTURE 10 1. Meaning of endosymbiosis, cyanobacteria, lateral gene transfer • Endosymbiosis: the theory that mitochondria and chloroplasts were derived billions of years aago from free living prokaryotic cells • Cyanobacteria: bacteria that can do oxygenic photosynthesis. They gave rise to the oxygen in the atmosphere • Lateral gene transfer: the transfer of genes between organisms in a manner other than traditional reproduction (eg. the relocation of genes in the organelle to the nucleus) 2. Origin of endomembrane system, nuclear membrane, ER etc. • The endomembrane system was derived from infolding of the plasma membrane • The nuclear envelope was an advantage by allowing the cell to regulate transcription and translation in a way that would not be possible without a nuclear membrane 3. Origin of mitochondria and chloroplasts • Mitochondria and chloroplasts were once free living bacteria that were incorporated into some larger bacteria and eventually became the organelles we see today 4. Evidence supporting theory of endosymbiosis • Mitochondria and chloroplasts have their own genome • Their morphology is similar to that of bacteria • They divide like a bacteria • They are not created de novo (mitochondria must divide for there to be more mitochondria) • They are the only parts of a eukaryotic cells that have electron transport chains • They have their own transcription and translation machinery 5. Factors driving development of early eukaryotic cells • Earliest bacteria were anaerobic but as the levels of oxygen in the atmosphere rose (due to the activity of cyanobacteria), cells could now do aerobic cellular respiration and create far more energy (in the form of ATP) than anaerobic bacteria could • The incorporation of mitochondria into cells allowed the cell to produce far more energy than it could on its own 6. Why eukaryotic cells can be larger and more complex than prokaryotic cells • Mitochondria allow the creation of far more ATP than could be possible without • ATP formation in bacteria is on their cellular membrane and as a cell increases its surface area (to try to get more energy) its volume also increases 2 • Volume increases at a greater rate (4/3 πr ) than surface area ( 4π r ) so a cell that can only do cellular respiration on its plasma membrane has to be small • Eukaryotic cells have far more energy due to mitochondrial activity so they can have larger, high energy genomes (lots of protein coding genes), they can have a low plasma membrane surface area to volume ratio (because the PM is not the site of oxidative phosphorylation), and lots of other cool stuff 7. Evidence for lateral gene transfer from organelles to the nucleus • The gene for complex 1 in the electron transport chain is found in the nucleus instead of the mitochondria where it is expressed 8. General idea about how lateral gene transfer is detected (Southern blot) • Lateral gene transfer is detected by searching for the presence of RNA by hybridizing a probe that anneals to its complementary strand • Mitochondrial DNA and genomic DNA is isolated from three related species and run on separate gels for the presence of a particular gene • Lateral gene transfer is still going on today so some species will have the gene present in mitochondrial DNA, some will have it in the genome, and some may also have it in both. This shows lateral gene transfer does occur and is still occurring 9. Hypotheses for why genes move to the nucleus from organelles (lateral gene transfer) • Over millions of years, genes have moved from the mitochondria to the nucleus to allow the nucleus to coordinate mitochondrial activity with the interests of the cell • This allows the metabolism of the cell to regulate mitochondrial activity • The mitochondria serves the cell instead of the other way around 10. Possible reasons why certain genes have NOT moved to the nucleus from organelles • Necessary gene products may not be able to be transported into the mitochondria • Localized gene expression may have advantages over transcription in the nucleus and translation in the cytoplasm then transport into the mitochondria • Some genes may be regulated within the mitochondrial environment 11. Role of cpn60 in tracing endosymbiotic and lateral gene transfer event in eukaryotes. • Eukaryotes that don’t have mitochondria (like giardia and trichomonas) have the gene cpn60 which is a mitochondrial protein and now found in the genome • This indicates that eukaryotes without mitochondria are not evolutionary intermediates, they had mitochondria and lost them (as evidenced by cpn60) • It seems the ancestor to all eukaryotes had to have mitochondria and that lateral gene transfer occurred very early on LECTURE 11 1. Relative sizes of typical mitochondrial, chloroplast and nuclear genomes • The nuclear genome has the largest genome by far (orders of magnitude bigger), the chloroplast has much fewer base pairs (about 200 kb) with the mitochondrion having the fewest base pairs (about 16 kb) • The typical prokaryote E. Coli has a much bigger genome than that of the organelles • This suggests that lateral gene transfer has occurred, making the genome in the organelles much smaller than expected for a typical prokaryotic cell 2. Rubisco structure and assembly from components coded by different genomes • Complex protein components are coded by different genomes (some in the chloroplasts, some in the nuclear genome) 3. Possible reasons why modern organelle genomes have become dramatically smaller over evolutionary time • Some genes are useful for free living bacteria but when mitochondria and chloroplasts were absorbed into the early eukaryote, some genes became redundant (like those for flagella and glycolysis) • Selection then favoured organelles with streamlined genomes because they are more efficient and use less energy to replicate and translate their genomes 4. Possible reasons why genes have moved to the nucleus from organelles over evolutionary time • Coordinated control between the nucleus and the organelles • Reactive oxygen species created in the mitochondria can harm the genome and cause mutations. You don’t want to keep your only library next to a fireworks factory • RNA can be edited in the nucleus though it cannot be edited in the organelles (no separation between DNA and ribosomes so translation directly after transcription) • DNA in the genome can be sexually recombined and generate diversity ( whereas organelles duplicate by binary fission) 5. Possible reasons why certain genes have not moved to the nucleus from organelles • Local control (some genes may be regulated within the mitochondrial environment) • Necessary gene products may not be able to be transported into the mitochondria • Localized gene expression may have advantages over transcription in the nucleus and translation in the cytoplasm then transport into the mitochondria • Some genes may be too big to move or cannot work in the nucleus (note the genetic environment in the organelles is prokaryotic and the environment in the nucleus is eukaryotic) • Not enough evolutionary time for gene movement 6. Basic mechanism of transcription and translation in prokaryotic organelles vs. eukaryotic nuclear environments • Eukaryotic nuclear environments are separated from the mechanisms of translation • This allows RNA to be modified in the nucleus before being translated in the cytoplasm (RNA editing, intron excision, etc.) • Prokaryotic genomes are within the cytoplasm and so are translated as soon as they are transcribed (no chance for editing) 7. Basic structure and function of RNA polymerase and ribosome • Ribosomes pair with proteins and must also interact with proteins • Ribosomes interact with tRNA and must understand mRNA • RNA polymerase is a protein that understands promoters (binds and begins to transcribe) and reads 3’ to 5’ (creating a complementary 5’ to 3’ RNA) • It also understand terminator sequences in prokaryotes (stop codons stop translation not transcription) • Ribosomes have a large ribosomal subunit and a small ribosomal subunit and have an E, P, and A site 8. Examples of complementary base pairing in gene expression • RNA pairs with itself to be able to function (mRNA, tRNA, rRNA, etc.) • Ribosomal RNA base pairs with itself to be catalytic • RNA pairs with other RNA (tRNA with mRNA, SD box and rRNA base pairing) • In DNA: in transcription, DNA base pairs with other DNA LECTURE 12 1. Identify the sequence of standard "start" and "stop" codons • The start codon is AUG in RNA, TAC in the template strand • The stop codon can be UAA, UGA, and UAG in the RNA 2. Identify the function of "start" and "stop" codons • Start codons are the signal for the start for translation, coding for the protein methionine. It is the first protein translated in mRNA • Stop codons do not code for any protein. A release factor binds to the ribosome when it encounters a stop codon and terminates translation 3. Compare the overall gene expression of prokaryotic vs. eukaryotic cells. • Prokaryotes translate the RNA as soon as it is transcribed • Eukaryotes separate the products of transcription in the nucleus from ribosomes in the cytoplasm so they have time to edit their RNA • Termination of transcription is different in eukaryotes and prokaryotes • Prokaryotes do not have introns or exons. Eukaryotes splice the mRNA to remove certain sequences identified in the cell as an intron • This allows one gene to code for multiple proteins by changing the sequences identified as introns and exons within different cells • In eukaryotes, RNA polymerase II cannot bind directly to DNA and so requires transcription factors to bind to the promoter before it can transcribe the DNA 1. Relative location of such DNA sequence “signals” as promoter, 5’ and 3’ UTR, “SD box”, start codon, stop codon, transcription terminator etc. • Promoters are not transcribed, they are located at the beginning of the transcribed gene and function to attract the RNA polymerase (along with a protein factor) • 5’ UTR (untranslated region) is the sequence before the start codon that does not get translated because it is upstream of the start codon (contains SD box in bacteria) • The 3’ UTR is the sequence after the stop codon (also not translated) • Transcription terminators are at the end of transcription. They are transcribed and loop to end transcription • The SD box is a region in the DNA (in the UTR) that when transcribed, base pairs with the rRNA and helps the initiation of translation (in prokaryotes) • Start codons are transcribed but are not the first bases in mRNA (preceded by the 5’ UTR and whatever else) • Stop codons are before the transcription terminator (and 3’ UTR) • Either strand can function as the template strand (depending on direction of promoter) 2. Mechanism by which each signal is interpreted, or understood, by the cell • Promoters are attractive to RNA polymerase and RNA polymerase creates a bubble of single stranded DNA. An A and T rich area precedes the start point (downstream where RNA transcription begins) • Start codons are understood by rRNA and interpreted as the place to start translation (by pairing with a Met tRNA) • Stop codons are understood by rRNA as the place to stop translation by attracting a protein in the A site that signals the end of translation (release factor) • The release factor is always trying to bind with the mRNA but is always outcompeted by the tRNA. When it comes to the stop codon, there is no tRNA to compete so the release factor binds and signals the end of translation • The transcription terminator is transcribed and base pairs with itself to form a hairpin loop. This loop signals to the polymerase to stop transcription (understood as RNA) • The SD box is a region in the DNA that when transcribed, base pairs with the rRNA and helps the initiation of translation (in prokaryotes) 3. Relationship between DNA sequence of signals and their function (ie. how would low efficiency promoters be different than high efficiency promoters?) • Promoters can be mutated to become more or less attractive to RNA polymerase • Stable binding means transcription is initiated more frequently • Terminators can also be mutated to be more or less efficient at stopping transcription (efficiency at base pairing with itself, less stable loops, longer loops) 4. Characteristics of promoters that require a particular position and direction • Either strand can function as the template strand so promoters must have a direction (i.e. they direct the RNA polymerase in a certain direction based on which strand they are located) • Where the promoter is located determines which strand is the template strand 5. Change in amino acid coded, given a change in the DNA sequence (and Genetic Code table) • Amino acids are sometimes coded by more than one codon (two or three or four) • If a DNA mutation occurs and the new codon codes for the same amino acid, that is a silent mutation (no change in overall protein) • If mutation occurs and the amino acid is changed, that is called a missense mutation (as in sickle cell anemia) • An amino acid can be changed to a premature stop codon, causing a nonsense mutation • A base pair may be inserted or deleted and so will shift the reading frame by one base pair (changing all the amino acids downstream of the in/del mutation) 6. Base sequence of start and stop codons as mRNA and DNA • Start is TAC in DNA template strands and so in ATG in the non-template strand. In mRNA, the start codon is AUG • Stop codons are UAA, UAG, and UGA in RNA. 7. The location of various signals given a diagram of gene expression • Starting from the 3’ end: promoter (not transcribed), 5’UTR (not translated), start codon, coding region, stop codon, transcription terminator (part of 3’UTR) LECTURE 13 1. Identify the main features of bacterial operons • A regulatory gene produces the repressor protein • An operator after the promoter but before the gene binds the lac repressor • A transcriptional unit containing the genes coded follows the operator 2. Identify the function of repressor proteins • Repressor proteins bind to the operator and inhibit transcription in certain conditions (i.e. the absence of lactose) • Changes in the cellular environment alters frequency of repressor protein binding and allow transcription of the gene (ex. allolactose binds to the lac repressor and alters its shape so it cannot bind). Lactose is an inducer • Thus, repressor proteins function to regulate transcription in response to changes in the environment (ex. presence of lactose) 3. Identify location of various components of the lac operon  The gene for the repressor is independent of the operon (regulatory genes can be located within an operon, adjacent to it, or far away). Called lacI  The lac repressor is located adjacent to the operon (according to pictures in the textbook)  The promoter binds RNA polymerase at the start of the gene and is followed by the operator (the promoter is not transcribed, the operator is, but neither are translated)  The transcriptional unit of lacZ, lacY, and lacA follows  Each have their own stop and start codons  The transcription termination site is last in the sequence 1. DNA signals in RNA-coding genes • Codons are coded 3’ to 5’ in the DNA but understood as 5’ to 3’ in mRNA 2. DNA sequence of anticodon in tRNA gene, given the codon • In DNA, Trp codon is ACC in the template strand. Therefore, the codon in mRNA is UGG. tRNA pairs with UGG (3’ to 5’) in mRNA so must have CCA anticodon (5’ to 3’). In DNA the anticodon is GGT (3’ to 5’) 3. Likely effect of base sequence substitutions in various DNA signals  Promoters and terminator sequences can become more or less efficient  Mutations to regulatory regions depends (not always bad or always good)  Start codons can only be broken (they are perfect at their job in the first place)  Stop codons can be mutated into another stop codon (no effect), or they can be broken and stop functioning as a stop codon (translation won’t work)  Many genes have redundant stop codons (to prevent effects of mutation)  Codons can be mutated in a variety of different ways, ranging from positive effects to no effect to varying shades of negative effects (bad to really, really bad) 4. Change in amino acid coded, given a change in the DNA sequence (and Genetic Code table)  Amino acids are sometimes coded by more than one codon (two or three or four)  If a DNA substitution mutation occurs and the new codon codes for the same amino acid, that is a silent mutation  If mutation occurs and the amino acid is changed, that is called a missense mutation (as in sickle cell anemia). Can be good, neutral, or bad  When a mutation occurs that turns the codon into a stop codon, translation is terminated too soon and this creates a nonsense mutation  A base pair may be inserted or deleted and so will shift the reading frame by one base pair (changing all the amino acids downstream of the in/del mutation)  Note: the start codon sets the frame (three possible reading frames) 5. Base sequence of start and stop codons as mRNA and DNA  Start is TAC in DNA template strands and so are ATG in the non-template strand. In mRNA, the start codon is AUG  Stop codons are UAA, UAG, and UGA in mRNA. ATT, ATC, and ACT in DNA 6. The location of various signals given a diagram of gene expression  In tRNA, there has to be a terminator sequence, an anticodon, and a promoter in the DNA sequence  tRNA is not a coding gene so they have none of the signals understood by translation in ribosomes (start/stop codons, protein coding region, etc.) 7. Basic structure of lac operon  Operons bring several genes under the control of one promoter 8. Mechanism of action of lac repressor  lacI is independent and has its own promoter. It produces the protein lac repressor  The lac repressor binds with the operator (through electrostatic attraction, not covalent bonds) and stabilizes a loop of double stranded DNA (not a hairpin loop) downstream of the operator  RNA polymerase cannot transcribe through the hairpin loop  Most of the time, the repressor is bound onto the operator 9. Function of lac operon in the presence, and absence, of lactose  In the presence of lactose, the lac repressor is inactivated (converted to an inactive form by the isomer allolactose) and transcription of the three genes is induced (b/c there is no repressor blocking transcription in the genome)  This is called a polysistronic message (one mRNA controls three genes) 10. Possible location of mutations in lac operon that give rise to a given phenotype  Mutation in the lac repressor gene could lead to inefficient binding, causing the lac operon to be transcribed more than needed  On the other hand, it could mutate to be insensitive to allolactase and the lac operon would be transcribed rarely or never  Mutations in the operator could make it incapable of binding lac repressor or it could be mutated to be far too attractive to lac repressor  Changes to the transcription unit could alter protein function  Changes to the promoter can lead to more efficient or inefficient binding of RNA polymerase 11. Phenotype that would arise from a given mutation in lac operon under given conditions  Increased binding of the lac repressor/inefficient promoter activity/alterations in coding sequence coyuld lead the cell to become unable to metabolize lactose  Decreased binding/increased promoter attractiveness can lead the proteins to be transcribed more often than needed and waste cellular resources  Alterations in the protein coding region can be neutral (silent mutation), damaging or helpful (missense mutation), or it can destroy protein function (nonsense mutation).  A frameshift mutation would likely affect every protein in the transcriptional unit LECTURE 14 1. Basic structure of eukaryotic vs prokaryotic cell with respect to gene expression  Eukaryotes have nuclear membranes so ribosomes are kept away from the mRNA 2. Structure of eukaryotic endomembrane system with respect to gene expression  In the nucleus, ribosomes are kept away from the mRNA  Allows RNA processing (posttranscriptional regulation)  Allows greater control of transcription  Tranlastional and post-translational regulation (alternative splicing) 3. Structure of eukaryotic promoters/enhanc
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