BIO 1002B TERM TEST 2LECTURE 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 membrane3. 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 today4. 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 machinery5. 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 own6. 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 Volume increases at a greater rate (4/3 r3) than surface area () 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 stuff7. 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 expressed8. 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 occurring9. 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 around10. 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 environment11. Role of cpn60 in tracing endosymbiotic and lateral gene transfer event in eukaryotes. Eukaryotes that dont 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 cell2. 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 genomes4. 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 dont 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 movement6. 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 site8. 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 DNALECTURE 121. 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 RNA2. 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 translation3. 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 strand5. 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), 5UTR (not translated), start codon, coding region, stop codon, transcription terminator (part of 3UTR)LECTURE 131. 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 operator2. 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 mRNA2. 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 wont 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 DNA6. 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 promoter8. 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 operator9. 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 polymerase11. 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 unitLECTURE 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 mRNA2. 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/enhancers Proximal regions are protein binding sites near the promoter. They are regulatory sequences (must be right next to the promoter) Enhancers can be quite far away from the gene that they regulate (do not have to be in one spot, positioned sort of independently) Inverting a promoter changes the direction of the gene completely but it doesnt matter for enhancers (not directional in the way promoters are) Enhancers bind to a multiprotein complex (coactivator) and the DNA loops to bring the enhancer-protein complex to the promoter proximal region This whole complex makes the promoter maximally attractive to the polymerase RNA polymerase II does not bind naked promoters. They have to be bound in the TATA box by TATA binding proteins Promoters are not as attractive unless they are bound by proteins in the TATA box. TATA binding proteins also attract additional transcription factors RNA polymerase II specializes in recognizing protein coding genes4. Protein motifs common in DNA binding proteins Proteins binding onto DNA to affect regulation is a common motif Proteins bind electrostatically. DNA is negatively charged along its backbone and proteins can be positively charged and attracted There are three kind of shapes that fit into DNA One is a helix turn helix DNA binding motif: one -helix binds to base pairs in the major groove of the DNA. A looped region of the protein (the turn) connects to a second -helix which holds the first helix in place Zinc finger DNA binding motifs are proteins with zinc co-factors. They form a particular shape that recognize DNA sequences Leucine zippers hold two monomer DNA binding proteins together (through hydrophobic interactions of the leucines). They are dimers consisting of -helical segments with other -helices binding to DNA base pairs in the major groove5. Mechanism of transcription termination in eukaryotes Transcription stops in eukaryotes due to the polyadenylation signal The polymerase transcribes right through the polyadenylation signal and that signal is recognized in RNA by a polyadenylase which cuts it The polymerase recognizes that the adenylase has cut the signal and stops transcribing The poly-A tail is then added on after transcription (no complementary base pairing). No poly-T6. Mechanism of translation initiation in eukaryotes Translation initiation begina as ribosomes scan for a start codon (no SD box) The small subunit recognizes the cap and slides along looking for the start codon7. Which gene expression components cross the nuclear membrane to get from where they are made to where they function Nuclear pores allow things to move in and out mRNA, rRNA, tRNA, miRNA all have to be made in the nucleus but have to function in the cytoplasm Nuclear proteins are synthesized in the cytosol but have to function in the nucleus8. Various stages of gene expression subject to regulation Transcription is the best way to regulate gene expression (no point making a message that is not needed) Cells can regulate transcription through protein factors like activators, coactivators, transcription factors, hormones, and methylation Introns are excised to regulate protein function in various cells mRNA can be masked by a protein (inactivating them)9. How organisms express different genes in each different tissue Unique combinations of activators control specific genes Some proteins may only be expressed in the eye, so only certain activators would be available in the eye vs. in the liver where different proteins are expressed (and so different activators are needed) miRNA can block certain genes in one kind of tissue and a different kind of miRNA is expressed and blocks expression of another kind of gene10. The advantages to alternative splicing Allows one gene to code for a variety of proteins which can be used in different, specialized tissue In some tissue, an intron is an exon where in different tissue it would be an intron Cells need to tell each other apart and to make the unique combinations of peptides needed to differentiate each other, they need alternative splicing You cant predict which sequence is an intron unless you see the end product11. Mechanism of action of miRNA Genes code for micro RNA They pair with themselves, then leave the nucleus and encounter a protein called dicer, which processes the RNA and the cut RNA associates with another protein forming a linear molecule of RNA in a protein complex The linear RNA is complementary to the 3 UTR of some other target mRNA Imperfect pairing of miRNA to sequence in target mRNA causes translation to be blocked (shuts down the expression of one particular gene)12. Mechanism of targeting proteins to cellular organelles Translation of all proteins begins in the cytoplasm Some proteins may be targeted for protein translational import via. channels They are a targeting tag that tells the cell to put the protein in the chloroplast, or in the ER (makes it rough), or in the nucleus, or whatever The tag is somewhere in the coding region of the DNA (since it needs to be coded and translated)13. Mechanisms to regulate protein function after they are made Amount of protein can be regulated by how quickly it is degraded The length of the polyA tail influences translation (longer tail means increased translation, shorter tail means decreased translation) Chemical modification, processing, and degradation14. Mechanism of ubiquitin/proteosome protein degradation Proteins are tagged with ubiquitin Proteosomes then degrades the protein15. The location of different types of information coded in DNA and how is each one understood by the cell Promoters and promoter proximal region are understood as DNA by activator proteins, transcription factors, co-activators, etc. and so regulate transcription The polyadenylation signal must be transcribed before it is recognized by polyadenylase and cleaved. Understood as RNA Start and stop codons are recognized by the ribosome during translation Codons are understood as the proteins the overall gene codes for. They are recognized by tRNA Enhancers are located in DNA and understood when coactivators bind to it and the promoter proximal region16. How prokaryotic gene structure/expression is different than eukaryotic Prokaryotes dont have introns, enhancers, polyA tails, or promoter proximal regions RNA polymerase in prokaryotes can bind directly to DNA17. Role of various types of RNA in gene expression snRNPs (protein and RNA complex) makes a spliceosome snRNA complementary base pairs with the message (introns contain a message in the DNA and are understood by the snRNP as the signal for cutting out the intron) This loops out the intron. This also allows for alternative splicing as different snRNA would be made for each intron needed to be cut18. Role of complementary base pairing in gene expression snRNA pairs with itself and with signals in the intron miRNA pairs with a target sequence and blocks translation19. Likely effect of mutations in various DNA signals Movement of the enhancer can make the gene less attractive If a virus comes in with a mega-enhancer the gene can be over expressedLECTURE 151. Identify various mechanisms for regulation of gene expression as summarized in Figure 14.6 Transcriptional regulation: chromatin remodeling, DNA methylation, availability of various activators, efficiency of enhancer/promoter, etc. Posttranscriptional regulation: variations in pre-mRNA processing like alternative splicing, RNA interference (through miRNA and siRNA), variation in mRNA breakdown (ex. through hormonal regulation) Translational regulation: miRNA silencing, and length of polyA tail Posttranslational regulation: variations in rate of protein degredation (ubiquitin), chemical modification of proteins2. Identify characteristics of genetic mosaics. Females with two X chromosomes inactivate most of the genes on one X chromosome or the other in most body cells For some genes, the inactivation of either X chromosome in heterozygotes produces different effects in distinct regions of the body For example, calico cats have orange and black patches of fur all over their body due to X inactivation (the allele for fur colour is on the X)3. Identify role of histones in DNA packaging and expression. Histones pack DNA molecules into the narrow confines of the cell nucleus Genes in regions that are tightly wound around histones are less active because their promoters are less accessible A nucleosome remodeling complex slides the nucleosome along to expose the promoter, thus regulating transcription Acetylation of the tails of the histones makes the histones less attracted to the DNA (by removing positive charges) and makes the promoter more assessable

1. Reasons why, if identical twin women have sons with identical twin men, the sons will not be identical. Their gametes will not be identical (they randomly assort and recombine) Random assortment of chromosomes in parents and recombination Different epigenetic markers2. Definition or explanation of "heritability" with respect to human disease risk. The variability in expression of disease risk due to genetic variation If you have the same genes as a person with schizophrenia you have a dramatically increased risk of getting the disease3. Characteristics that would, and would not, be different between monozygotic twins Gene expression, different gametes Environmental mutagens, natural mutations in their cells X inactivation, levels of DNA methylation4. Process of random X inactivation leading to genetic mosaicism. Mammalian females are mosaics Random X chromosome inactivation creates genetic mosaic In early embryogenesis, each cell will inactivate one of its Xs randomly Half of the cells will have the maternal X active and half will have the paternal X active (all descendants of each cell will keep the same X inactive) Two different cell lines from the same germ line5. How a mosaic is different from a heterozygote. Heterozygotes have both genes active, a mosaic inactivates one gene6. Role of Xist RNA in X inactivation. Xist RNA binds along the inactive X chromosome, silencing most genes It tightly condenses the chromosome by coating the chromosome and keeps the DNA away from transcription factories (not complementary base pairing)7. Role of Tsix RNA in regulation of Xist RNA expression. Xist and tsix genes are transcribed in opposite directions at the same locus One at the top strand, one at the bottom strand (spelled backwards) Tsix shuts off the expression of Xist8. Meaning of "antisense". The opposite strand (backwards to other strand)9. Structure of nucleosomes. Nucleosome: DNA wouhnd around core of 2 molecules of four different histones DNA is wrapped twice around the histones10. Function of nucleosomes in chromatic structure. Nucleosomes coil DNA into solenoid fibers DNA becomes very tightly packed and hard to express11. Role of nucleosomes in chromatin remodeling. Nucleosomes can be moved, to remodel chromatin near promoters Genes can be shut down or freed up to influence gene expression12. Relationship between acetylation/deacetylation of histone tails on chromatin structure. Histone tails can be modified to affect DNA packaging Acetylation of histones opens up the chromatin and increases gene expression Deacetylation deactivates gene expression13. Relationship between methylation/demethylation of cytosine on chromatin structure or protein binding. DNA methylation can prevent binding of a transcription factor Methylation of DNA might attract the deacetylase enzymes which will then compact the chromatin14. Factors that may influence epigenetic methylation patterns in DNA. Diet, exposure to toxin, chemicals, maternal care15. Difference between "genetic" vs. "epigenetic" changes that affect gene expression. Genetic changes influence what genes you have available in the first place Epigenetic changes influence what genes are turned on and off Methylation is stable and lasts a lifetime Epigenetic changes can be heritable across generationsLECTURE 161. Identify the function of caspases. Destroys the cell in response to activation of the trigger for programmed cell death

1. Likelihood that modern multi-cellular life forms are monophyletic. All data supports the hypothesis that multicellularity evolved several time Many groups have some multicellularity and some unicellularity Volvox genus is not monophyletic (evolved communal lifestyle multiple times independently)2. Characteristics of Volvocine algae that make them a useful model system for studying the transition to multi-cellularity. Volvocine algea consist of somatic cells and reproductive cells (differentiated) There are many cells that display varying levels of cellularity, from completely unicellular to spherical colonies with a communal cell wall to differentiated cells The divergence of Volvox and Chlamy is quite recent (50 million years to most recent common ancestor)3. Relative structure/function of Chlamydomonas vs. Volvox cells. Chlamy is completely unicellular and the cells are undifferentiated Volvox is a sphere of cells full of ECM (reproductive and somatic)4. General process of Volvox asexual reproduction; role of somatic vs gonidial cells. Gonidial cells divide multiple times. Some cells become larger than others (asymmetric division) and the large cells become reproductive cells Smaller cells become somatic cells At some stage, the cell is inside out (flagella on the inside, gonidial cells on the outside) and so the cell inverts itself The gonidial cells leave the main cell at some point to form a new colony5. Genetic approaches to identifying genes relevant in rise/maintenance of multi-cellularity. Mutating a group of cells and examining the mutants that cannot form multicellular colonies to see which genes might be affected Compare genome of Chlamy and Volvox Studying gene expression somatic and reproductive cells6. Types of data or insight revealed by comparative genomic studies in Chlamydomonas vs. Volvox Protein coding genes are identical in size Genomes are pretty much the same. Developmental innovation did not require a particularly complex genome Two exceptions: more cyclins (control cell cycling) and more genes for cell wall7. Types of data or insight revealed by mutation/rescue experiments in Volvox. Three genes important Gls seems responsible for the asymmetric division (shifts plane of mitotic division) Lag gene represses the flagella and the eyespot genes and whatever else is not needed for reproduction RegA represses reproduction in somatic cells8. Difference between orthologous and paralogous genes. Orthologue: the gene in Chlamy and the gene in Volvox are derived from one common ancestor (orthologous) Orthologues are the same gene, but repurposed (new function) Paralogue: a related gene has a different regulation Ex. a gene in the common ancestor caused cells to divide less in response to low light That gene duplicated and in Volvox this gene has a different regulation (influences development of somatic cells)9. Types of genetic changes associated with multi-cellular growth in Volvox. Genes can be the same as in Chlamy but repurposed Genes can be related but have a different method of regulatory LECTURE 171. Identify the various mechanisms for regulating the activity of mRNA after they are transcribed Masking mRNA, RNA interference (through miRNA and siRNA), variation in mRNA breakdown (ex. through hormonal regulation) RNA interference is carried out by noncoding single-stranded DNA that bind to mRNA and affect their translation miRNA in a protein complex binds to mRNA and stops translation. It does not destroy it if the pairing is imperfect miRNAs control gene expression in developmental processes by turning on and off gene copies in response to the timing of development siRNA is produced from double stranded RNA not encoded by nuclear genes siRNA-induced silencing complex targets the RNA and it is cleaved and degraded, not simply silenced for a short time (used to protect against viral attacks)2. Identify the various mechanisms for regulating the activity of proteins after they are translated. Proteins can be tagged for degradation by ubiquitin, tagging for transport to various organelles, feedback regulation of transcription (protein product binds to promoter and stops further transcription), conversion of inactive protein precursors to active form, addition or removal of chemical groups

1. Typical causes of cell death. Predation (virus or bacteria), toxins, mutational load, programmed cell death2. Mechanism of plasmid toxin/antitoxin system as a possible origin for cell death genes. Bacteria suffer from infection by parasites (which are just little bits of DNA) The infectious plasmids replicate themselves and send themselves to another cell The recipient cell does not want the plasmid. Cells that divide and lose the plasmid have an evolutionary advantage so over time the population could lose the plasmid The plasmid doesnt want that so it produces toxins and anti-toxins The toxin is long lasting but the anti-toxin is quickly degraded Cells that lose the plasmid are killed by toxin So the cells build the anti-toxin gene into their genomes and the plasmids respond by making an anti-toxin toxin and so on It is hypothesized that these genes were later put under developmental regulation so cells can activate the toxins for programmed cell death3. Programmed cell death cascade in C. elegans. CED-3 is a caspase (a protease that digests particular proteins) CED-4 activates CED-3 CED-9 is a mitochondrial protein that regulates CED-4 A death signal begins a cascade, activating CED-9 which releases CED-3, which activated CED-3, a caspase that activates proteases and nucleases4. Possible evolutionary origin of programmed cell death genes. The anti-toxin/toxin system was taken over by the cell and put under developmental control. Cells can commit suicide when they need to When cells took up mitochondria, they also took up the mechanism of cell death Apoptosis is intimately associated with mitochondria5. Characteristics that make Drosophila an attractive model system. Theyre small, theyre not considered sentient (no need for ethics), short lifespan6. Main stages in Drosophila embryonic development. An egg is fertilized and nuclear division begins Nuclear division without cellular division creates a bag of nuclei which then migrate to the poles of the cell They grow cell membranes and become separate cells (blastoderm) The blastoderm segments and grows into an adult The larva turns into a pupa which turns into the adult fly7. Main role of maternal effect, segmentation and homeotic genes in Drosophila development. Mothers pack their eggs with information that the zygote is going to need after fertilization (embryonic genes take a while to be expressed) The eggs get packed full of proteins and mRNA which will be useful immediately after fertilization. But the mRNA must be prevented from being translated until fertilization so the mRNA is masked Bicoid mRNA is only expressed near the head (because thats where the mRNA is packed). This is a gradient of gene expression. Maternal effect genes determine polarity Segmentation genes: gap genes subdivide the embryo into segments Each segment turn on the next set of genes called homeotic genes Homeotic genes determine structures and location Gene duplication and divergence over time can give rise to increasing complexity by allowing different homeotic genes to be expressed in different segments8. Structure/function of the "homeobox" in homeotic genes These genes contain a signal called the homeobox because the protein made from this homeobox signal is the one that binds onto the promoters of target genes The homeobox is a helix-turn-helix DNA binding domain It allows these proteins to regulate a whole bunch of other genes Several genes under the same transcription factor allow several proteins to be under the control of one thing Hox genes are evolutionarily conserved (highly conserved set of developmental homeotic genes). Present in fruit flies and mice and humans and so on9. Role of programmed cell death in Drosophila development Programmed cell death destroys larval tissues not needed as an adult Ecdysone stimulates programmed cell death Imaginal disks are little pouches of cells that just sit in the larva and wait until the larva has eaten enough. Then a blast of hormones (ecdysone) transforms the worm into a fly The upstream region of Reaper protein has binding sites for ecdysone, p53 (indicating DNA damage), and Hox genesInformation in DNA: codons, promoters, terminator sequences, start codons, stop codons, SD box, methylation