MBS 350M/388M Fall 2024 Exam 1 - PDF

MBS 350M/388M Fall 2024 EXAM ONE (LECTURES 1-7) Lectures 2 and associated textbook readings 1. Understand the big range in nuclear genome sizes, including in plants. Note that nuclear genome size means the haploid nuclear genome size in base pair (bp), kilo base pa...

MBS 350M/388M Fall 2024 EXAM ONE (LECTURES 1-7) Lectures 2 and associated textbook readings 1. Understand the big range in nuclear genome sizes, including in plants. Note that nuclear genome size means the haploid nuclear genome size in base pair (bp), kilo base pair (kb, this is 1000 bp) and so on to higher units. 2. What are the main features of the DNA sequences that contribute or have not much effect to having such a wide size range? Added note to clarify: In question above, identify what are types of DNA (can be more than one) that are most common as factors for the large variation in eukaryotic genome sizes. Features that vary and contribute to the wide range of nuclear genome sizes 1. Amount (or fraction) that is highly repeated 2. Abundance of other DNA (transposons, etc.) 3. Frequency and sizes of introns a. Humans have very large introns 4. Genetic redundancy (or duplication) a. Fraction of genes that are in gene families, and the size of the families 5. Horizontal gene transfer (from different species) Among eukaryotes, # of genes (protein coding, non-protein coding, like rRNAs) does not track closely with genome size. 3. Briefly, how are chromatin fibers compacted to help pack large amounts of DNA (chromatin) in the nucleus? Sophia- Traditionally, Chromatin fibers are compacted by wrapping DNA around protein complexes called nucleosomes, forming a bead on string structure which coils into a 30 nm fiber. The 10 nm fiber coils into a compact 30 nm fiber which is the primary level. Then the 30 nm fibers loop and fold upon themselves, condensing the chromatin into a final chromosome structure. chromatin are a bunch of nucleosomes put together 4. Define polyploidy in contrast to haploid and diploid. How do groups of cells in multicellular plants become polyploid in certain developmental cases? Added note to clarify: In question above, what change(s) occur in the cell cycle so that diploid cells become polyploid? Generally, how may the polyploid state of these cells benefit the organism? Sophia- 1. Haploids- have one set of chromosome (example: sperm and egg) 2. Diploid- 2 sets of chromosomes (example: from mother or father) 3. Polyploidy- 3 or more sets of chromosomes - in plants, increased Ploidy in whole organisms or select cells can increase size of Nuclei, cell and organs. - Endoreduplication: common in plants (continuous DNA replication) - example: 2n -4n (DNA is replicated but cells don't undergo mitosis and cytokinesis. 5. What are some cellular and physiological effects of having whole genome polyploidy conditions in plants? Ploidy effects in Arabidopsis: large flowers and more branched leaf trichomes ○ Compensatory regulation: cell sizes increase with ploidy almost linearly and Organ size increase is more modest due to reduced cell number (not proportional) Lecture 3 and associated textbook readings 1. What cellular processes can give rise to polyploid plants (undergone whole genome duplication) naturally? Induction of polyploidy in commercial plant breeding Natural breeding producing polyploidy (errors, mitosis, meiosis) are too infrequent for breeding High temp. Not very effective Better option: Chemical inhibitors of mitosis such as colchicine. Widely performed. could be naturally caused by aneuploidy: The occurrence of one or more extra or missing chromosomes in a cell or organism. when pairs of chromosomes don't complete the process of cell division and fail to separate. 2. Depending on the species, can tetraploids cross with diploids and produce viable generations of offspring that are hybrids? Understand the cellular processes affecting these outcomes to the extent we covered in lecture. Do major crops have a range of ploidies? What is necessary to maintain a triploid crop? Understand several ways in which polyploid organisms can affect evolution in ways that diploid organisms are more constrained. Yes. Cross the produces triploid offspring and problem with gamete formation Start with a diploid and tetraploid (diploid undergoes meiosis to become haploid and tetraploid undergoes meiosis to become diploid) and then together the haploid and diploid become an F1 triploid (three copies of each chromosome) crops can also have a range of ploidies triploids are usually sterile and infertile, and tetraploids are usually fertile 3. What are some major ways in which genomes become varied (mutated) that may affect the many functions of the genome such as gene expression (production of proteins and RNAs), replication etc)? Some sources of genome variation - Base pair substitutions - Insertions and deletions - Inversions and translocations - Gene duplications - Chemical inhibitors of mitosis such as colchicine. Widely performed. 4. How can genes associated with certain traits be identified? Once genes are identified, what is marker-assisted selection in general terms? Can you explain why marker-assisted selection will be faster, more efficient than genetic trait selection in a breeding program? The Chapter 2 readings and Lecture 3, last two slides can help you understand this. Use of genetic variation to generate superior crops -a method to advance is marker assisted selection or marker assisted breeding EX: In rice, problem of flooding and crop submergence for days at a time A rice variety was found with better submergence resistance 1. Traditional Breeding: cross w/ modern cultivar and test for the desired trait in progeny , successive back-crossings with the modern cultivar. 2. Marker assisted breeding: same but identify and clone the gene for trait or a nearby gene, then use PCR to quickly follow presence of the gene in the Progeny. a. reduces the amount of time required for identification of different breeds that have the desired trait in a breeding program. Lecture 4 and associated textbook readings Note: Like to highlight that Chapter 3 of text is very good on transposons 1. Understand the two major types of transposons in plants and how their transposition mechanisms differ to the extent we covered in class. What type of transposons are better known for moving? Retrotransposons: copy and paste through RNA intermediate, increases # of transposons. DNA elements/ transposons: cut and paste, # of transposons stays the same. Retrotransposons are considered more mobile: the intermediate stage in retrotransposition allows for multiple copies of the transposons to be generated through reverse transcription, leading to a higher potential for rapid amplification and spread throughout the genome. 2. Can you describe the trend in TE content and genome size in plants? The linear relationship between the log of the genome size and the amount of TES in the graph on the right shows that most of the difference in size is due to the difference in TE content. 3. Understand autonomous and nonautonomous members of a transposon family. How do these terms describe the famous Ac/Ds transposon system in maize and their interaction? What type of transposons are the Ac/Ds transposons? autonomous:TE can move on its own within the genome because it contains all the necessary genetic information (including the transposase gene) to perform transposition nonautonomous: refers to a transposon that lacks the ability to move independently and requires the presence of a functional transposase encoded by an autonomous element within the same family to facilitate its movement AC/Ds are DNA transposons "Ac" stands for "Activator" and is considered an autonomous transposon, meaning it can move on its own, while "Ds" stands for "Dissociation" and is a non-autonomous transposon, requiring the presence of the Ac element's transposase enzyme to move; essentially, Ac provides the machinery needed for both its own movement and the movement of Ds elements within the genome. 4. What are 2-3 factors which limit transposon movement to the extent we covered in class? Sophia- 1. movement depends on autonomous transposon expression of the gene (they can only physically relocate within the genome) 2. TES become particularly localized in areas of heterochromatin (centromeres and telomeres 3. TEs are subject to gene silencing by DNA methylation, histone modifications and RNA dependent silencing 5. What are 2-3 ways transposons can affect other genes’ expression? Include a positive and negative outcome in general terms. negative: if transposons insert themselves into genes it can induce mutations or disrupted genomes Positive impact: transposon insertion can introduce new regulatory elements, like promoter sequences, near a gene, leading to increased gene expression under specific conditions. Lectures 5 and associated textbook readings 1. You should be familiar with nucleosome composition and structure, that there is packing of them to form higher chromatin structure but no need to learn specific packing structures. refer back lecture 2 number 3. 2. Define telomeres. You should be familiar with telomerase structure and activity, how it enables the maintenance of chromosome length to the extent we covered in lecture. It is not necessary to sketch the actual mechanism. Telomeres ○ Repeated dna at the ends of eukaryotic chromosomes ○ Nucleoprotein structures that cap the ends of eukaryotic chromosomes  Telomerase has an associated RNA that complements the 3’ overhang at the end of the chromosome. ○ RNA template is used to synthesize the complementary strand ○ Telomerase shifts and the process is repeated ○ Primase and DNA polymerase synthesize the complementary strand. ○ telomerase adds telomeres to ends to prevent degradation 3. Describe in general terms the chromosome end structure and how this protects chromosomes from damage. telomeres protect chromosomes from damage by preventing chromosome degradation(it puts a “cap” on the chromosome ends to prevent damage using proteins and stuff. Specifically , without this complex, the ds (double stranded) DNA ends would be recognized as cut DNA that needs to be repaired. Result that ends of one chromosome may be joined to ends of another chromosome causing massive genome disruption. 4. How can many plant species continue to grow and make offspring through flowers even after many years, with bristlecone pines being at the amazing extreme in longevity to be able to do this? Understand how plant telomeres and telomerase expression with aging can be different from animals in the somatic and meristematic (stem) cells. Length range for telomeres (few 1000 p to 150 kb in different species Grow continually flexible delivery In Arabidopsis, meristm had longer telomeres then differential cells (same applies to some other plan species not unique to arabidopsis (constant regeneration) In arabidopsis, differential cells can regenerate into new organs including flowers. 5. Define centromeres. What are CENH3 proteins and how do they affect nucleosome structure and function in centromeres? Centromeres: - Site of chromatid attachment and kinetochore attachment - Usually one per chromosome but can be numerous - Usually heterochromatin but hac transcribed RNAS low rate of recombination - In plants, centromere DNA often consists of unique to species repeats, LTR(long terminal) retrotransposons and few genes. - Histone variant for H# called CENH3 (CENP-A in mammals) present in centromeres of plants, fungi, widely. Hypothesized 3D structure of a rice centromere CENH3 containing nucleosomes have distinctive differences from H3 containing nucleosomes At the centromere, CENH3 containing nucleosomes have binding affinity for one or more proteins that make up the kinetochore, hence kinetochores assemble at the centromeres 6. Be aware that haploid plants can be produced by crosses where one parent has mutant less functioning CENH3 proteins. Why does this happen? Why are haploid plants helpful for plant breeding? Production of haploids by chromosome elimination to aid basic studies and plant breeding Mutated CENH3 containing nucleosomes binds proteins to build kinetochore less efficiently than wild type Female parents with mutated CENH3 genes can make some haploid gametes. But in first zygotic divisions, the female inherited chromosomes will not build kinetochores well and youtube some haploid cells only with paternal chromosomes, can make haploid plants Lecture 6 and associated textbook readings 1. Understand the endosymbiotic origin of chloroplasts and mitochondria. Are archaea or bacteria considered to be more likely as the cell type which did the original engulfing? What type of bacteria gave rise to chloroplasts? Understand that the red lineage of algae and protists happened due to secondary endosymbiosis. Chloroplast Origins and evolution The cp genome is fairly conserved in evolution compared to nuclear or mitochondrial genomes Originated from endosymbiotic associations Endosymbiotic hypothesis- most associated with lynn margulis It postulated that the primary endosymbiont was a cyanobacterial-like organism. Therefore, Cp DNA is a remnant of that cyanobacterial genome, and most of its other genes were either lost or transferred to the nucleus. - Primary endosymbiosis: the original internalization of prokaryotes by an ancestral eukaryotic cell, resulting in the formation of the mitochondria and chloroplasts. - Secondary Endosymbiosis: a process that occurs when a eukaryotic cell engulfs another eukaryotic cell that has already undergone primary endosymbiosis. This process results in the formation of a cell with organelles from two different lineages, and chloroplasts with more than two membranes. 2. Did the genomes of the endosymbionts stay largely intact through evolution? If not, what changes happened generally? Describe in general terms the chloroplast and mitochondrial genomes----physical structure as circular and/or linear genomes and be able to list a few processes that organelle encoded gene products participate in, in each organelle. they did not stay intact: the changes that occurred were genome reduction, dependence on the Host, lost of redundant pathways (meaning they no longer have the genes to synthesize molecules, and lose important functions because the host provides essential amino acids/vitamins) Chloroplast Genetics: Inheritance is typically, Uniparental (usually maternal) Multiple mechanisms involved, not well understood Chlamydomonas: the parental (-) cpDNA is destroyed and the maternal (+) cpDNA is replicated In some land plants, the parental plastids are excluded during fertilization or absent from the sperm cell 2. Essentially, all plastids have DNA, usually the same DNA throughout the Organism (homoplasy: a rare evolutionary occurrence in which unrelated organisms independently develop the same trait) 3. The DNA sequence does not change during differentiation (except sometimes in variegation: the presence of different colored zones in a plant's leaves, stems, or fruit.) - note: there are exceptions to the last two statements. Digestion of cpDNA of the maternal parent in a young zygote of Chlamydomonas revealed by fluorescence staining of DNA In a young Chlamydomonas zygote, fluorescence staining of DNA can reveal the digestion of chloroplast DNA (cpDNA) from the "minus" mating type parent, demonstrating the phenomenon of uniparental inheritance where only the cpDNA from the "plus" mating type parent is retained, effectively showing the degradation of the maternal cpDNA in the zygote. Chloroplast DNA General features: Double stranding, circular molecule No histones, but has other bound proteins (example: Hu) organized into nucleoids Prokaryotic features like Operons, one RNA pol, encodes prokaryotic size rRNAs. Multiple copies (approximately 30-100) per plastid (for instance, all chloroplast genes are multi-copy) Can be 10-20% of the total DNA in leaves (or more than 1000 copies per cell) Mitochondria Structure: Outer membrane Inner membrane Intermembrane space Matrix Plant MT DNA & genetics circular , usually (not always) Example: Chlamydomonas and related green algae have linear mt DNA, similar to protists. Some higher plants have a mix of linear circular. More variable in size and structure than chloroplast DNA No histones Low copy number per organelle (melon leaf cells had 30-80 copies per organelles) Inherited uniparentally (but not always) Conifers: from both parents Angiosperms: Maternal (same as cpDNA) Chlamydomonas: from the mt(-) parent (cp DNA inherited from the mt(+) parent) Cp = Chloroplast 3. How and when are the organelles replicated, compared to the cell mitotic cycle? How are the organelle genomes generally inherited when gametes fuse to make a zygote? Organelles are primarily replicated during the G2 phase of the cell cycle, which occurs just before mitosis, ensuring that each new daughter cell receives a sufficient number of functional organelles; this replication happens through a process of organelle division, where existing organelles simply split into two, with the newly formed organelles being distributed roughly equally between the daughter cells during cytokinesis. Mechanism: Organelles like mitochondria and chloroplasts replicate by dividing into two identical copies through a process called binary fission, where they simply split apart. Inheritance in gametes: When gametes fuse to form a zygote, organellar genomes are typically inherited primarily from the female gamete (egg) due to the larger amount of cytoplasm it contributes, leading to a phenomenon called "maternal inheritance". 4. What are proplastids and where do they mainly exist in the life cycle of land plants? Can you mention 2 differentiated plastid types, how they differ structure and function, be sure to include chloroplasts. Proplastids are small, undifferentiated plastids found mainly in the meristematic cells (actively dividing cells) of plants, such as those in roots, shoots, and developing seeds. These organelles serve as precursors to various specialized plastid types. During the plant’s development, proplastids can differentiate into other types of plastids depending on the environmental cues and developmental signals the cell receives. Plastid Type Chloroplasts Double membrane, internal Photosynthesis, energy production, thylakoid membrane system with synthesis of fatty acids and amino acids chlorophyll. and pigments for and immune response roles. photosynthesis Chromoplasts Double membrane, lacks Pigment synthesis and storage, thylakoids, contains pigment attracting pollinators and seed granules (carotenoids). dispersers, photoprotection. Leucoplasts Double membrane, lacks pigments and Storage of starch (amyloplasts), thylakoids. Subtypes: Amyloplasts, lipids (elaioplasts), or proteins Elaioplasts, Proteinoplasts. (proteinoplasts). 5. Are there multiple chloroplasts and mitochondria per cell? Be aware of the wide range from single chloroplasts in unicellular algae to hundreds in leaf cells while mitochondria also have a big range but usually hundreds. Yes, due to secondary endosymbiosis Lecture 7 1. Why do chloroplasts & nuclei need to communicate and coordinate? Please note that same applies—there is mitochondria and nuclei coordination. Need for chloroplast and nuclear communication and coordination: - chloroplasts have several 1000 proteins but on;y encode usually less than 100 proteins - Some multi-subunits proteins like ATP synthase have both Chloroplast and nuclear encoded subunits - Dynamic changes in protein levels during development or in response to environmental conditions. Mechanisms of coordination: Signaling from the nucleus to chloroplast resulting in chloroplast response Signaling from the chloroplast to the nucleus resulting in nuclear gene regulation (retrograde signaling) 2. What is chloroplast-nuclei retrograde signaling? Where does the signal originate and where does it travel through to where there is a response to the signal? Are the signals known to be large molecules like proteins or RNA or small metabolites? Signaling from the chloroplast to the nucleus resulting in nuclear gene regulation (retrograde signaling)  ○ the signal- outside signals, photosynthetic signals inform about status. 3. To help you see an example of retrograde signaling, are you able to sketch out the steps for the effects of high light stimulus and the response in the nucleus? Be able to mention 2 other stimuli which initiate retrograde signaling to the nucleus, understand that they involve different molecules and have different gene expression effects; no need to learn the detailed steps in these other pathways we covered. High Light - (HL)- bright light intensities High Light stimulus Excess electrons beyond capacity of photosystems created singlet oxygen 1O^2 Singlet oxygen oxidation of carotene to make beta-CC in chloroplast Crosses chloroplast membranes through postulated not unknown transporter In cytosol, beta-CC may activate a protein which in turn transmit signal to nucleus Signal selectively induces antioxidant protective genes in Nucleus. Biotic stress Stimulus (pathogen or insect) Stimulated isoprenoid pathway in the chloroplast MEcPP is specific regulatory intermediate MEcPP crosses chloroplast membranes (unknown transporter) to cytosol, moves into nucleus Selectively induce pathogen resistance genes in the nucleus possibly by chromatin remodeling. Binds to known transcription factors activates stress response genes. High light + drought stimulus (also heat is a stimulus) PAP level increases in the chloroplast PAPST1 is likely pap transporter across chloroplast membrane Crosses cytosol postulated to move into nucleus via nuclear pores Inhibits major XRN ribonuclease family from degrading mRNAS and miRNAS Increased mrnas and mirnas for enhanced abiotic stress responses and stress tolerance. (process is fast) 4. In the study of maize seedlings described in Lecture 7, what was the unique regulation the researchers found for the psbA mRNA which encodes D1 protein that was not observed for other mRNAs on polysomes during light-dark transitions? Why may this observed regulation be beneficial to the plant? Story: Most mrna occupancy on polyribosomes unchanged only psbA mrna occupancy higher in light. In light, increased translation all mrnas D1 protein synthesis is especially high in light needed to replace damaged D1 protein. Maize plants in diurnal light to dark and vice versa measured chloroplast ribosome occupancy by mrnas and translation rates Most mrna occupancy on polyribosomes unchanged only psbA mrna occupancy higher in light. In light, increased translation all mrnas D1 protein synthesis is especially high in light needed to replace damaged D1 protein. Maize plants in diurnal light to dark and vice versa measured chloroplast ribosome occupancy by mrnas and translation rates Method to measure mrna occupancy on polyribosomes Isolate polyribosomes Treat with ribonuclease, then purify ribosome protected mRNA fragments Make cDBA copies and sequence cDNAs to identify which and how much of a mRNA is on polysomes Ribosome profiling: Functions of core translation initiation factors Upstream translation and alternative start sites Elongation speed and ribosome pausing Cotranslational folding and localization Termination recycling, and quality control Translation regulation by microRNAs and RNA-binding proteins.  EXAM TWO (LECTURES 8-?) Lectures 8 and associated textbook readings 1. Be able to define retrograde signaling and how it can be of physiological benefit to the organism. In animals and plants, what typically is the general change in mitochondria which initiates retrograde signaling? As presented in lecture, can plant mitochondria affect chloroplast retrograde signaling to the nucleus? 2. Understand what RNA editing is and its importance. Be able to list a few of the eukaryotic groups exhibiting RNA editing. In which organelle(s) does it occur in plants (not all plants show editing)? What type of nuclear encoded protein is involved in RNA editing and how is editing so precisely executed? Give the type of protein(s) but it is not necessary to remember the specific name. Textbook Chapter 5 is particularly good on RNA editing. 3. What is trans-splicing and why is it important? In plants, where in the cell does trans-splicing take place? We discussed a nuclear mutant in rice that encodes an RNA binding protein. This mutant has less trans-splicing of one of the mitochondrial electron transport chain carriers. 4. What is the function of alternative oxidase in plant mitochondria? Are these enzymes found in mammalian mitochondria? When there is a defect in the electron transport chain in plant mitochondria (such as in the rice mutant in Point 3) or when there is a developmental regulation in flowers such as the corpse plant, how does alternative oxidase protein levels and enzyme activity vary in the mitochondria? For these two examples, be able to state how altered alternative oxidase activity may benefit the plants. Lectures 9 and 10 and associated textbook readings 1. Have an introductory biology level of understanding tRNAs, rRNAs, mRNAs, their functions. Also be aware of non-coding RNAs (nc-RNAs, these are regulatory non-coding) and several types of small RNAs (no need to learn specific names now). 2. Understand secondary and tertiary structures of RNAs can be critical to function. Such as in ribozymes and riboswitches, good to have idea of how these work. 3. Gene organization and features: generally, how are nuclear genes spaced and transcribed along the duplex DNA? Understand the general layout of proximal promoter elements, transcription start site, introns, termination sequences. 4. Understand that there are 5 nuclear RNA polymerases in plants, of which RNA pol IV and V are unique to plants, that they transcribe different types of genes, the resulting transcripts can be processed extensively. These general points are good except just remember that RNA pol II synthesizes mRNAs. The organelles have 1-few RNA polymerases with similarities to prokaryotic enzymes. All these RNA polymerases use DNA as template. 5. Can the evolution of eukaryotic RNA polymerases I-III from prokaryotic RNA polymerases still be detected from the protein structures? 6. RNA pol II uniquely has a C-terminal domain (CTD) among other RNA polymerases on one of the big subunit(s). What are the distinctive protein features of the CTD? How does the CTD generally become modified during the full start to finish transcription cycle as we discussed and what are the functional effects of these modifications? What types of proteins carry out the modifications? 7. Can you describe events and components which enable RNA pol II to be correctly bound at the core promoter (that is, form pre-initiation complex)? It is not necessary to know the names of the general transcription factors (GTFs) and how many are used. Just good to learn the initial TFIID with the TATA-binding protein subunit is first to bind core promoter. Are there TATA-less promoters that TFIID binds to? When RNA pol II is bound by GTFs only, is there a high or low rate of transcription? 8. For a higher rate of transcription, how do enhancers or proximal promoters with bound activating transcription factors (not repressive transcription factors) stimulate higher rates of transcription? Note the roles of protein:protein interactions including the build-up of a Mediator complex to stabilize the pre-initiation complex. How is RNA pol II released from the pre-initiation complex at the core promoter to start moving? Lectures 11 and associated textbook readings 1. Be able to name the typical multiple domains found on specific transcription factors (TFs). Include the function(s) of each domain. 2. Be aware there are multiple families of transcription factors in eukaryotes based on their protein structures, some are unique to plants. It is not necessary to remember the names of the families we covered in class: bHLH, bZIP (also called Leucine zipper) and zinc finger families. What is common among the DNA binding domains that helps them bind the DNA? Some TF families exist as homodimers and heterodimers----what do these descriptions mean and what expanded functions can heterodimers provide compared to homodimers? 3. For DNA sequences bound by TFs, about how many base pairs typically constitute a DNA binding site? In general terms, how does the TF:DNA interaction achieve specificity so that the correct TF binds the correct gene regulatory sequence? 4. Be familiar with the main steps in chromatin immunoprecipitation (ChiP) as a commonly used method to identify TF binding sites. Main steps to remember: first a chemical is used to cross-link proteins bound to DNA, then that lysate is incubated with an antibody that recognizes the TF of interest, the TF-DNA-antibody complex is purified based on the antibody, then DNA is released. How is the DNA usually analyzed? 5. We’ll use enhancer and proximal promoter descriptions for where selected specific TFs bind DNA as these terms are still widely used. Good to understand generally where these are relative to the transcription start site. Can there be multiple DNA binding sites and multiple TFs at each location? 6. How do bound specific TFs stimulate transcription initiation via their effects on Mediator complex? (Not necessary to recall the parts of the Mediator complex, that in process, a kinase subunit must be removed from Mediator before it can interact with the pre-initiation complex). Lecture 10 also covered this. Lectures 12 and associated textbook readings 1. Post-translational modifications of TFs. For each of the ones we covered--- phosphorylation, SUMOylation, ubiquitination----what do these terms refer to and what are the general cycles affecting their activity and what are some ways they affect TFs? Include the basal non-stimulated condition, what happens when a stimulus is present, and how does the system reset to basal condition when the stimulus is no longer present. 2. Learn about one specific TF we covered in lecture—either the Arabidopsis ABI5 TF or the Arabidopsis ICE1 TF. How is it regulated according to the activation and return to basal conditions processes? Include the physiological stimulus, the proteins that directly affect the TF abundance or activity (not necessary to include the steps prior to the protein directly acting on TF in signaling pathway) and generally, what types of genes does the TF regulate when it is activated (example cold stimulus will affect genes that increase cold tolerance). 3. TFs can move in the cell from cytoplasm to nucleus and back to cytoplasm. In the case of membrane bound TFs generally, where are they located in basal conditions and how are they processed to enable entry into the nucleus? Comment on how this system may enhance control of TF activity. 4. Be aware that a regulatory protein(s) can interact with a TF complex affecting complex formation or activity. Regulatory proteins and TFs can move cell to cell through plasmodesmata. 5. What is crosstalk in the condition when an organism experiences multiple stimuli at the same time affecting gene transcription? How can crosstalk benefit the organism? Lecture 13 parts 1 & 2 and associated textbook readings 1. RNA-directed DNA Methylation (RdDM) is a process unique to plants and uses two plant-specific RNA polymerases. Understand the steps and molecules used to compact chromatin, repress transcription of TEs and repeat DNAs as presented in class. 2. Non-coding RNA regulation example: control of flowering by period of cold (vernalization). Understand how FLC gene is repressed to relieve the FLC transcription factor mediated repression of flowering. Begin with cold and COLDAIR non-coding RNA made across the 1st intron of FLC gene and end with FLC gene in repressed state, including the steps in the process. Lecture 15 will expand to show several additional regulations. 3. siRNA (and miRNA) transport: understand transport via plasmodesmata cell to cell and the use of extracellular vesicles (exosomes) to move RNAs out of one plant cell into extracellular space and into another plant cell (or other cell like fungal pathogen). Lecture 14 and associated textbook readings 1. Can you describe the general layout of a nucleosome? What are the proteins and where is the DNA? How do the N-terminal tails on histones differ from the structure of the rest of the histone proteins when histones are assembled into nucleosomes? 2. How do acetyl groups on histones affect the structure and function of the chromatin? What are the enzymes which add and remove acetyl groups from histones? Which amino acid did we discuss as the main modified amino acid? How are HATs localized to specific gene sites and then how can they be involved to affect chromatin propagated outwards from an initial binding site? 3. How do methyl groups on histones affect the structure and function of the chromatin? What are the enzymes which add and remove methyl groups from histones? Which amino acid did we discuss as the main modified amino acid? How are HMTs localized to specific gene sites? Hint, can be TFs or recall that HMTs can be part of PRC2; other mechanisms we discussed. Be familiar with a couple of these. 4. How do chromatin remodelers enable formation of the pre-initiation complex for start of transcription? Just understand the basic ATP-dependent chromatin remodeler mechanism affecting nucleosomes, no need to distinguish pullers and pushers. NOTE: not necessary to recall HAT interactions with Mediator subunits nor the insulator/boundary proteins limiting epigenetic spread. Lectures 15 and 16 1. For DNA methylation, which base can be methylated? Understand that the base must exist within a short DNA sequence motif. What are some mechanisms which will guide DNA methyltransferases to methylate or not methylate a given DNA sequence in the chromosome? How is the DNA methylation state stably maintained? How does DNA methylation repress or activate transcription? Please be aware that methyl groups can be removed (not necessary to remember the mechanism of several enzymes that are used to demethylate DNA). 2. Lectures 15 and 16 brought in case studies of epigenetic regulations in plant development (expanded information on FLC gene regulation) and plant host-pathogen interactions. Some great biology to share with you! Enjoy! I will not ask questions on the midterm specifically about these presented case studies, that is the information about them from these lectures. Lecture 17 and 18 and associated textbook readings---note these are chapters 11, 13, and 14. Please draw from both lectures for learning these points as the two lectures had overlapping material. 1. 5’ Cap on mRNA: Briefly, describe what is the cap and its location on the mRNA (no need to draw structure. Recall from the RNA Pol II transcription and Pol II CTD connection discussed earlier that the cap is added shortly after transcription initiates. What are 2-3 of its valuable functions? Be able to describe at least one function in the nucleus including nuclear export and one function in the cytoplasm. 2. 3’ end cleavage and polyadenylation: Similarly to above for cap, recall the RNA Pol II CTD connection and the transcription stage these processes happen. In plants, these processes takes place differently from other eukaryotes. In plants, where on the mRNA from a given gene does 3’ end cleavage and polyadenylation take place? How do the resulting mRNAs differ? Can you think of how 3’ end differences may affect regulation of the mRNAs? For poly A tails, what are 2-3 of its valuable functions? Be able to describe at least one function in the nucleus including nuclear export and one function in the cytoplasm. 3. Alternative splicing: understand how it differs from regular splicing in general terms and how it can increase protein diversity. To help you understand how this alternative splicing can occur, include your understanding that an RNA binding protein can block the splicing machinery to skip an exon as an example. 4. Understand the steps in miRNA biogenesis beginning with miRNA gene transcription in the nucleus and ending with the miRNA as part of RISC in the cytoplasm to the extent covered in lecture. Making a sketch, labeled with annotations, is my strong recommendation. About how big is a miRNA? What is the function of the 3’end methyl group on miRNAs in plants? What is a RISC composed of and in a few words in your sketch annotation, how is the miRNA (guide strand) identified for retention while the other strand (passenger strand) is ejected or degraded from the pre-RISC? 5. When a miRNA binds to a target mRNA, what two effects are possible for the effects on the mRNA? In your sketch, include as presented in lecture, that a miRNA will be bound by a particular AGO. When this RISC binds mRNA, it in turn recruits effector proteins (no details on their names) to accomplish one or the other effects on the mRNA. Understand general point that a given miRNA can regulate multiple target RNAs as long as there is sufficient base pairing between an mRNA and a RISC so that expression of many genes can be regulated by the miRNA. 6. What processes determine the level of an mRNA in a cell? How can varying mRNA half-lives or turnover rates benefit the cell and organism as distinct from transcriptional control of gene expression? 7. For RNA binding proteins, are there regulatory post-translational modifications? is reversibility built in to the regulatory system? Be able to name 2-3 of the modifications as presented in lecture. In general terms so not specific to a given modification, how can these modifications affect RNA binding protein activities such as RNA binding, role in translation or physical state such as are tagged for protein degradation. 8. For RNA modification, be aware that reversible modifications such as methyl groups can be added to RNA bases and these have regulatory effects (no details on this mechanism). Lecture 19 and associated textbook readings 1. What are Stress Granules and Processing Bodies and their general functions? Note that RNA binding proteins in the cytoplasm are one mechanism for transport of a specific mRNA to one of these complexes (see point 2 also). 2. We used macrophages and their regulation on cytokine mRNA levels as a great example to illustrate RNA binding protein-mRNA interactions leading to degradation of specific mRNAs. No need to know the details but good to understand generally that RNA binding proteins can bind specific sequences or secondary structures in the target mRNAs including in the 3’-UTR. Once bound, can you list 2 ways they can promote mRNA degradation? 3. Understand that eukaryotic cytosolic translation is divided into initiation, elongation and termination stages. We focused on initiation because it is generally the most regulated step. 4. What are the structures and interactions that lead to circularization of mRNA during translation? How does this arrangement benefit initiation and the overall process of translation? 5. What is the definition of translational efficiency for a given mRNA? What are features of mRNA 5’UTRs that affect translational efficiency? Note that different mRNAs can have highly differing translational efficiencies due to these differences. 6. How can translational efficiency vary depending on development, stress and other conditions so that translational efficiency of an entire or almost entire population of cytoplasmic mRNAs is affected? In these cases, how are such broad effects achieved (give molecular mechanisms)? 7. What is the connection, if any, between translation on polysomes and mRNA degradation process? Lecture 20 and associated textbook readings 1. For protein folding, understand basic structures (motifs) found in proteins: alpha helices, beta sheets and what type of bond occurs between cysteines? Understand that chaperones aid in protein folding to their functional structure states. 2. How are proteins sorted to particular cell compartments or secreted? Be familiar with the general idea of protein sorting (signal) sequences, that they are short sequences on the proteins, can occur in different places along the protein length depending on the type of signal. 3. For endomembrane system targeting, understand that the signal peptide is bound by SRP and there is co-translational transfer of the protein from the cytosol into the rough ER membrane or lumen. Specific other steps are not needed. 4. How are proteins transferred from one membrane bound organelle in the endomembrane system to another, or alternatively to the plasma membrane, or secreted out of the cell? Just describe in general terms about the use of receptor proteins, you will not be asked to diagram. 5. For chloroplast targeting, be able to describe the process beginning with finished protein in cytoplasm and its intended location in the chloroplast, giving steps and components in this process. No need to learn the specific names of proteins and the translocation complexes; can just refer to as their general names such as chaperone, protease and translocation complexes. For mitochondrial protein targeting, just understand has similarities and differences from the chloroplast system. Lecture 21 and associated textbook readings 1. Be able to mention 1-2 places where programmed cell death takes place in life cycle of plants. Note that there are several different terms used for entire cell death besides programmed cell death like apoptosis, but we’ll use programmed cell death. 2. Understand that there are different catalytic types of proteases (diff enzymatic mechanisms) and they can occur in different cell compartments. 3. Good to learn the widespread process of the ubiquitin-proteasome system (UPS). Be able to briefly describe the process of 3 enzymes are used to attach ubiquitins to target proteins and how proteasomes act on the targeted proteins as described in lecture. Good to remember name and function of the third enzyme: E3 ligase; the other enzymes can be just described simply by what they do. Where do proteasomes occur in the plant cell? 4. How does high light intensity damage Photosystem II (PSII) chloroplast proteins? Include the general role of Reactive oxygen species (ROS). What does D1 protein do in photosynthesis? Understand and be able to diagram the main events of replacing damaged D1 protein, beginning with damage and ending with restoration of functional D1 and PSII. You can use general terms for steps and proteins involved to the extent we covered in lecture, no need to have the specific names of proteins active in the process. 5. The Gibberellic acid (GA) signal pathway, be able to mention 2-3 physiological processes regulated by this hormone. Be able to describe one GA signaling pathway (either the PIF example or the JAZ example), which uses UPS to extent we covered in lecture. Begin with low [GA] condition then cover high [GA] condition ending with positive or negative effects on transcription, giving the molecular steps/molecules along the way. The only names good to specifically show in your description are GID1, DELLA while rest can be more general like TF, Ubiquitin conjugating enzyme (or E3 ligase), transcription factor (example, PIF) or transcription repressor (example, JAZ). Lecture 22 (no textbook readings for this) 1. Good to refresh and understand how PCR works, the enzyme and steps involved to produce a large amount of DNA corresponding to the DNA between the PCR primers. Gel electrophoresis and staining the DNA so it can be visualized is one way to detect the PCR product(s) from a PCR reaction. 2. Molecular understanding of how CRISPR/Cas9 system produces gene inactivation as a genetic engineering method—you can use Lecture 22, slide 10 to help you see the steps. Note these are simplifications, but ok for basic understanding. Going over the natural CRISPR Cas system in bacteria and archae can help your understanding but this is optional. 3. Understand how CRISPR/Cas9 system creating modifications with the same species DNA is currently not considered a GMO plant. How are GMO plants defined? 4. We covered two examples of metabolic engineering in crops in some detail. You can focus on either one to become familiar to answer the relevant question below. -Why has the team of scientists chosen to alter glycolate metabolism as a way to increase photosynthesis and crop growth? -Why has the team of scientists chosen to use elevated suberin production in crops as a way to draw down atmospheric CO2? 5. In area of Ecosystem stewardship, please remember well the bottom lines: 1) High species diversity generally is good, creates a more stable ecosystem. 2) Genetic diversity within a species is essential so that the species can better adapt, survive in changing conditions, Molecular biology can help monitor both of these as one tool for stewardship. 3) Genetically selected or created lines can help restore species in trouble in the ecosystem to better health and fitness.