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Lecture 22 “Cell-Cell Interactions: Communication between Adjacent Cells” PPT Review: 1.) What general forces does the extracellular matrix of cells protect against? a. 1.) Cross-linked network of filaments protects against stretching forces 2.) Stiff ground substance protects against compression 2.) What is the primary cell wall composed of? What type of macromolecule is this? How is it packaged and arranged in the cell wall? What molecule fills the space between this cell wall material and what type of macromolecule is this? a. Primary cell wall composed of cellulose b. Carbohydrate (polysaccharide) c. Consists of long cellulose strands bundled together into structures called microfibrils (fibers) d. Space between these is composed of Pectin—a carbohydrate (polysaccharide) 3.) What material makes up the “cross-linked filaments” of plant cell ECM? Ground substance? a. Cross-linked filaments = cellulose microfibrils b. Ground substance = Pectin 4.) What is turgor pressure? a. The force exerted against the plant cell wall caused by increasing cell volume (could be due to water uptake) 5.) In regards to composition, what is the difference between plant ECM and animal ECM? What makes up the fibrous component of animal ECM? What type of macromolecule is this? What makes up the matrix that surrounds the fibrous component? a. Animal ECM contains much more protein relative to carbohydrate than does a cell wall b. Collagen makes up the fibrous component c. Collagen is a protein d. Matrix is composed of gel-forming proteoglycans 6.) Where are most ECM components synthesized in animal cells? Where are they processed? How are they secreted from the cell? a. Rough ER b. Processed in Golgi c. Secreted via exocytosis 7.) What will dictate variation in both the amount of and composition of ECM in animals? a. Location—the tissue type of the cells will cause it to adopt different composition or a varying amount of ECM (p. 203 in text for more) 8.) Why are integrins an important protein for cell-cell interaction? a. Integrins are extracellular proteins that bind to other proteins that connect to the cytoskeleton. Therefore, these proteins form a linkage between the ECM and cytoskeleton which helps adjacent cells adhere to each other 9.) What is the example we discussed that demonstrates one possibility of cells losing their cellECM connection a. Cancer metastasis—increased cell motility 10.) Describe tight junctions. If a line of cells connected by tight junctions were to come in contact with a solution, how would the solution cross beyond the barrier of cells?—Why is this important? a. Tight junctions allow cells to form a watertight seal with cells adjacent to them. b. Tight junctions force solution to instead pass through the cells rather than past them. c. ^^Therefore, the cells can regulate or restrict movement of substances between the cells and the rest of the body which is why these cell connections are important in locations such as the intestines or skin layers. 11.) What is selective adhesion? What type of cell adhesion proteins did we discuss in lecture? Where are these proteins found? What is their significance? a. Cells of the same tissue type and species (of cell) have specific adhesion molecules that allow them to adhere to each other b. Cadherins c. Found in Plasma membranes d. Cadherins only bind to cadherins of the same type. Therefore, they allow cells of the same tissue type to attach specifically to one another. 12.) Adjacent cells have specializes proteins that assemble in their membranes which form structures that allow a channel to form and thus serve as communication portals. What is the name of these structures in animal cells? What are they in plant cells? What type of molecules would pass through these channels? a. Gap junctions in animal cells b. Plasmodesmata in plant cells c. They admit water, ions, and small molecules—AA, CHOs, and nucleotides Lecture 23 “How Cells Communicate: Communication between Distant Cells” PPT Review 1.) What is a hormone? Examples of hormones from PPT? a. Hormone is an information-carrying molecule that are secreted by plant and animal cells into bodily fluids and act on distant target cells b. Insulin, nitric oxide, estrogen, auxin 2.) Describe signal receptors and how they respond to their corresponding ligand. How do signal receptors serve as a control mechanism for determining the functionality of cells? a. Signal receptor changes its shape and activity after binding to a signal-molecule b. Specific signal receptors are present only in specific cell types—the book provides the example that a signal for dehydration (and to therefore conserve water) can be distributed to the whole body, however only kidney cells will respond because only they have the specific receptor that binds to the molecule that relays the “message” 3.) Explain why there are both intracellular and extracellular receptors. a. This can be explained by the signaling molecule’s ability to pass through the membrane (think back to selective permeability and hydrophobicity). If a signaling molecule can diffuse through the membrane into the cytosol, their corresponding receptors will be present as intracellular receptors—if not, extracellular receptors. 4.) If the signaling molecular is lipid-insoluble, how is the signal transmitted to the cell? What are the two types of receptors for lipid-insoluble molecules? 5.) 6.) 7.) 8.) 9.) a. Signal transduction pathway which will convert the extracellular signal to an intracellular signal b. G protein coupled receptors (GPCRs) and Enzyme-linked receptors Outline the general steps of a signal transduction pathway. a. Ligand (Primary messenger) binds to extracellular receptor --> Binding causes a signaling cascade which releases second messengers --> A cellular response is triggered or a molecule translocates to the nucleus and causes a change in gene expression Outline the steps of the GPCR signaling pathway. a. G-protein is inactive b. Signal (ligand) binds to receptor spanning the PM c. Receptor-ligand interaction causes GPCR to change shape and activate the G-protein d. Activated G-protein results from bound GDP being exchanged for GTP e. G-protein undergoes conformational change causing the active portion of the G-protein (with GTP bound) to break off from other subunits of the G-protein f. Activated G protein binds to an enzyme which leads to the production of second messengers to then lead to downstream cellular responses (depending on what the signaling molecule-GPCR’s functionality entails) Explain the significance of primary signaling messengers leading to secondary messengers? a. Through signal transduction pathways, a single primary signaling messenger can lead to an abundance of secondary messengers being released—think “signal amplification” Outline the steps of an enzyme-linked receptor signaling pathway (the book uses a receptor tyrosine kinase (RTK) pathway to describe this. a. Signal binds to receptor b. Receptors dimerize (receptors are now active) c. Receptors cross-phosphorylate/auto-phosphorylate using ATP inside the cell d. Proteins inside the cell (‘bridging proteins’ on the PPT) bind to receptor to form bridge to Ras (a G-protein) e. Ras exchanges GDP out and brings GTP in (activated Ras) f. Phosphorylation + activation of protein kinase (we talked about mitogen activated protein kinases (MAPK)) g. Phosphorylation cascade (the protein kinases phosphorylate each other) h. Response triggered in the cell (such as gene activation in nucleus) Keep in mind that signal deactivation is crucial for the cell to continue proper functioning. Based on the types of enzymes present in the RTK pathway example, what type of enzyme could interact with the pathway to largely deactivate the signal transduction? a. Phosphatase protein—could counteract the activity of the kinases in the phosphorylation cascade Lecture 24 “Control of Cell Cycle” PPT review: 1.) Watch the Mitosis video from lecture. What are the different phases of mitosis? What happens at each phase? a. Interphase—after chromosome replication, each chromosome is composed of two sister chromatids. Centrosomes have replicated b. Prophase—Chromosome condense and spindle apparatus begins to form c. Prometaphase—Nuclear envelope breaks down. Microtubules contact chromosomes at kinetochores. d. Metaphase—Chromosomes complete migration to middle of cell e. Anaphase—Sister chromatids separate into daughter chromosomes and are pulled to opposite poles by spindle apparatus f. Telophase—The nuclear envelope re-forms, and chromosomes de-condense 2.) What are the phases of the cell cycle? What occurs during Gap phases? Explain the G0 phase. a. G1 growth to accommodate new genetic info—organelle replication (and in G2) etc b. S phase—DNA synthesis c. G2—constructs microtubules, prepares mitotic proteins, cytoplasm growth etc 3.) Define the following: a. MPF—M-phase promoting factor = a complex of a cyclin and CDK that when activated will phosphorylate a number of proteins needed to initiate mitosis in eukaryotic cells b. Cdk—cycling dependent kinase = protein kinase that is functional only when bound to a cyclin and are activated by other modifications. c. Cyclin = regulatory protein whose concentrations fluctuate cyclically throughout the cell cycle. Involved in control of cell cycle via cdk. 4.) How are Cdks activated? Explain the accumulation and degradation of cyclin in cells. a. Has to bind to cyclin b. Cyclin concentration increases during interphase and peaks in M phase, where it is then destroyed 5.) What is the relationship between concentrations of cyclin and activity of MPF? a. When cyclin concentrations are high, more MPF is active 6.) What is the function of Cdk? (include the reaction it’s involved in) a. Cdk is a protein kinase. It phosphorylates protein targets. 7.) There are 3 cell cycle checkpoints we discussed in lecture: a. What occurs at each checkpoint/What is being “checked”? i. G2—Checking if the DNA is both replicated and undamaged ii. M-Phase—Checking if chromosomes have attached to the spindle apparatus and that the chromosomes have segregated properly iii. G1—Checking if the cell is large enough, has enough nutrition to proceed through the rest of the CC, and that the DNA is undamaged b. State whether MPF is present or absent. i. G2—MPF present ii. M-phase—MPF absent 8.) Explain how the G1 checkpoint is subject to social control using the slides from lecture or book. What is acting as the “social control” in this? 1. Growth factors arriving from other cells (increased [growth factor]) and stimulate the production of E2F and G1 cyclins 2. Rb binds to E2F, inactivating it. G1 cyclins form cyclin-Cdk dimers. The dimers are phosphorylated which inactivates them 3. The inactivating phosphate on the cyclin-Cdk dimer is removed 4. Active Cdk phosphorylates Rb 5. Phosphorylated Rb releases E2F 6. E2F stimulates production of S-phase proteins a. The growth factors (keep in mind these are from other cells though) are acting as social signals triggering the Rb protein to be overridden—allowing E2F to help progress the cell to S-phase 9.) What characterizes cancer on a cellular level? a. Uncontrolled cell growth, accumulation of genetic changes 10.) If a cancer cell divides without growth factors, which checkpoint does it bypass? Explain why cancer cells passing through this checkpoint is a problem—think about what occurs at the phase following G1. a. G1 phase b. Problem because G1 checkpoints are checking for DNA damage (among other things). Therefore, if these checkpoints are not operating properly and the cell passes through unchecked, DNA damage will be present in the DNA when replicated. 11.) During what phases in the cell cycle would you expect there to be large changes in the polymerization or depolymerization of microtubules? a. Polymerization—while the mitotic spindle is forming—prophase b. Depolymerization--Anaphase 12.) When actively growing cells are treated with Taxol, they often are unable to complete the cell cycle. Based on what you have learned about cell-cycle checkpoints, which checkpoint likely causes these cells to arrest? a. Taxol inhibits cell cycle at Metaphase of M-phase Lecture 25/26 “How Genes Work” Review: 1.) What is a gene? What is gene expression? a. A section of DNA that encodes information for building one or more related polypeptides or functional RNA molecules along with the regulatory sequences required for its transcription (Text) b. The set of processes, including transcription and translation that convert information in DNA into a product of a gene, most commonly a protein (Text) 2.) What is the “one gene, one enzyme” hypothesis? a. The hypothesis that each gene is responsible for making one enzyme (Text). i. But was adapted to include genes that produce RNA the final product b. A particular stretch of DNA (a gene) contains the information to specify the amino acid sequence of one protein (Lecture PPT) 3.) What are examples of genes encoding for functional RNA as final product? a. tRNA, ribozymes, etc. 4.) What is the triplet code in molecular biology/what is a codon? Why is it considered redundant? a. A code in which a “word” of three letters encodes one piece of information. The genetic code is a triplet code because a codon is three nucleotides long and encodes one amino acid (Text) b. Redundant because all amino acids except M and W are coded by more than one codon 5.) What are the start codons? What are the stop codons? a. Start = AUG (Met) b. Stop = UAA, UAG, UGA 6.) What is the central dogma of molecular biology? a. DNA RNA Protein i. DNA transcribed to RNA translated to protein 7.) How did RNA viruses “update the central dogma”? a. RNA viruses have the capability of synthesizing DNA from an RNA template by utilizing an enzyme called reverse transcriptase. So: RNA DNA 8.) What determines an organism’s genotype? What is phenotype? Do different alleles of a gene differ in DNA sequence? a. Genotype is determined by the sequence of bases in the DNA b. Phenotype is the physical trait that is a product of the proteins produced c. Yes, they differ in sequence—we used the example of the melanocortin receptor (mc1r) in class i. The two alleles differed in only one AA but the phenotypic differences were drastic 9.) Define each (and be able to recognize/replicate each on your own): a. Point Mutation—A single base change in DNA b. Missense Mutation—A point mutation that changes one AA in in the AA sequence (so a single mutation in a codon—resulting in the resulting AA to be altered) c. Silent Mutation—A point mutation that does not change the AA sequence (redundancy with the triplet code makes this possible) d. Nonsense Mutation—A point mutation that changes an AA-coding codon to a stop codon e. Frameshift Mutation—Addition or deletion of nucleotide that causes the reading frame to shift (likely resulting in drastic change to the AA sequence) Lecture 27 “Transcription of Genes: Production of RNA” Review: 1.) What is the function of RNA Polymerase? What is the template strand and what is the coding strand? What direction does RNA Polymerase perform its template-directed synthesis (strand polarity here)? a. RNA Pol synthesizes an RNA transcript using one strand of the DNA b. Template strand is the DNA strand that is read by the RNA Pol. The coding strand is not transcribed but its sequence will match that of the transcribed strand (aside from the uracil/thymine difference) c. RNA Polymerase transcribes in the 5’ 3’ direction 2.) Does RNA Polymerase require a primer? What is the name for the region of DNA that RNA Polymerases interact with during transcription initiation? a. RNA Polymerase doesn’t require a primer b. The gene promoter (sequence) 3.) What are the components that make up the bacterial RNA Polymerase Holoenzyme? What is the function of each component? a. The core (RNA Polymerase) enzyme and Sigma b. RNA Polymerase catalyzes the RNA synthesis reaction while Sigma regulates transcription initiation 4.) What is the significance of the -35 box, -10 box, and +1 box? In bacteria, what component of the RNA Polymerase holoenzyme interacts with the DNA initially during transcription initiation? Where does the component bind and how is this assisting the RNA Pol core enzyme? a. -35 and -10 box contain sequences that commonly occur as promoter regions. The numbers indicate how far they are upstream from the starting point of transcription— the +1 box. b. The sigma factor binds to the -35 and -10 boxes and orients the enzyme at the start site 5.) Once the holoenzyme is bound to the DNA, what change must occur in the DNA helix in order for RNA Pol to transcribe a single RNA strand? What enzyme causes this change? a. RNA Polymerase will open the DNA helix which will create two separated DNA strands, allowing the template strand to pass through the active site of RNA Pol 6.) Is the reaction catalyzed by RNA Polymerase exergonic or endergonic? Why? a. The reaction is exergonic due to the potential energy stored in the 3 phosphate groups of the NTPs—think back to the repulsive forces of the negative charges present between the phosphates in the triphosphate group 7.) What is the step that ends initiation? What is the name of the second step? What is happening during this step?(—think about what the RNA Pol is physically doing during this step) a. RNA Polymerase extends small mRNA from the +1 site b. Elongation c. Active site of RNA Pol is catalyzing the addition of nucleotides to the 3’ end of the RNA 8.) What is the final step in bacterial transcription? What causes this to occur? What happens to the orientation of the RNA molecule immediately after this final step? a. Termination b. Transcription is terminated when RNA Polymerase transcribes a DNA sequence that functions as a transcription-termination signal. c. Once the termination signal is transcribed into RNA, this portion of the RNA forms a hairpin which disrupts the interaction between RNA Polymerase and the RNA transcript—this separates the two 9.) What is the RNA Polymerase that transcribes protein-coding genes in eukaryotes? How does the promoter sequence differ in eukaryotes compared to bacteria? What proteins in eukaryotes serve the same function as sigma factor did for bacteria? How does termination of eukaryotic protein-coding genes differ from bacteria? a. RNA Polymerase II b. Eukaryotic cell promoters are highly variable but many contain a TATA box upstream of the start site c. Basal transcription factors d. Termination involves the poly(A) signal—after the signal is transcribed the RNA is cut downstream of the Poly(A) signal 10.) What are exons? What are introns? Does the primary RNA transcript contain both exons and introns? a. Exons = Sequences that ligated after excision—these are present in the final mRNA product b. Introns = Sequences that remain physically separated after excision—these are sections of genes that are not represented in the final RNA product c. Primary RNA transcript does contain both exons and introns 11.) What are the proteins that make up the spliceosome? What is the function of the spliceosome? a. Small nuclear ribonucleoproteins (snRNPs) b. The spliceosome removes the introns, allowing the exons to ligate together 12.) During RNA processing, what must occur to the 5’ end and the 3’ ends of the RNA transcript? What is the function of both of these processing steps? a. 5’ cap added—a modified guanine with 3 phosphate groups and a Poly(A) tail added to the 3’ end—consists of 100-250 adenine nucleotides b. 5’ cap serves as recognition signal for the translation machinery. The 3’ Poly(A) tail protects the mRNA from degradation Lecture 28 “Translation of mRNA: Protein Synthesis & Post Translation Modifications” Review: 1.) What is the site of translation in both eukaryotes and bacteria? What does it mean to say that transcription and translation are tightly coupled? Are they coupled in both bacteria and eukaryotes? Explain why. a. Ribosomes b. If coupled, translation will occur directly after (or even while) the mRNA is completed c. Transcription and translation are coupled in bacteria but not in eukaryotes d. The DNA is in the cytoplasm of bacteria. Therefore, the transcribed genes are immediately within the cytoplasm as well (as mRNA), which allows the ribosomes to interact with them. DNA of eukaryotes is in the nucleus and is separated from the 2.) 3.) 4.) 5.) 6.) 7.) 8.) 9.) cytoplasm. This allows for more control of when the resulting mRNA will be expressed in eukaryotes. What is the function of a polyribosome? a. Allows many copies of a protein to be produced from a single mRNA What facilitates the interaction between amino acids and mRNA in ribosomes? Within the ‘facilitator’, what type of structure does the AA interact with? What does the mRNA interact with? Think back to RNAs with tertiary structure. a. Transfer RNA (tRNA) acts as an adapter between the two b. The AA interacts with a specific sequence (CCA) at one end of the tRNA c. The mRNA interacts with anticodon at single-stranded portion at a different end What catalyzes the addition of amino acids to the tRNA? How is the molecule able to do this? (What within its structure allows it and what is it recognizing). a. Aminoacyl-tRNA synthetase b. Each aminoacyl-tRNA synthetase has a binding site for a particular AA and a particular tRNA. Subtle differences in tRNA shape and base sequence allow it to recognize the correct tRNA for the correct AA What is the term used to describe a tRNA molecule covalently linked to an AA? a. Aminoacyl tRNA What is the wobble hypothesis? What paradox does it resolve? a. Wobble hypothesis states that an anticodon can form nonstandard base pairing (at the third position) with AAs that have redundancy in their codons i. This is explained by the fact that many AA are specified by more than one codon and codons for the same AA tend to have the same nucleotides at the first and second position but different only at third— 1. The book uses example that CAA and CAG both code for Glutamine. So the resulting tRNA has anticodon (GUU) that matches for CAA but for CAG it has nonstandard pair at the third position (U paired with G) b. It resolves the paradox that there are 61 different mRNA codes but most cells only contain 40 tRNA Where in the cell are ribosomes assembled? What are the substructures of the ribosomes and what are their basic functions? a. Nucleolus b. Small subunit holds the mRNA in place during translation | Large subunit is where peptide-bond formation occurs What are the three sites in ribosomes that tRNA occupies during translation? What are the basic interactions occurring at each? 1. A-site—the acceptor site; site that the aminoacyl tRNA initially makes contact with the codon 2. P-site—the peptide-bond formation site; site that holds the tRNA with growing polypeptide 3. E-site—the exit site; site that holds a tRNA (that contains no AA anymore) to be released What are the steps of translation initiation (from the PPT)? 1. mRNA binds to small ribosomal subunit 2. f-MET tRNA (the initiator aminoacyl tRNA) binds to small ribosomal subunit 3. Large subunit then binds Elongation can now proceed 10.) What are the steps of translation elongation? 1. Incoming aminoacyl tRNA moves into the A site (the f-MET tRNA has moved into the P-site at this point) 2. Peptide-bond formation between the new AA (held by the tRNA in the A site) and the f-MET 3. Translocation of the f-MET tRNA into the E-site and the new tRNA into the P-site. This leaves the A-site open for the next incoming aminoacyl tRNA 11.) What type of enzyme is a ribosome? What part(s) of the ribosome is/are composed of rRNA? a. Ribosome is a ribozyme (catalyzes protein synthesis) b. rRNA forms the catalytic center for peptide bond formation, decoding site, intersubunit interface, A, P, and E sites. 12.) What are the steps in translation termination? a. Once the ribosome reaches a stop codon, a release factor binds to stop codon at A site b. Polypeptide and uncharged tRNAs are released c. Ribosome subunits separate 13.) What is the primary difference between the release factor and the aminoacyl tRNA that bind before it? How does the binding of the release factor cause the separation of the polypeptide from the ribosome? a. The protein release factor does not carry an amino acid b. The active site of the protein catalyzes the hydrolysis of the bond that links the tRNA in the p-site to the polypeptide. By doing so, the polypeptide is not bound to anything anymore and is therefore released. 14.) Why might a protein need to undergo a post-translational modification? What are posttranslational modifications that we’ve discussed in lecture? a. PTMs often activate some proteins or target them to specific locations b. Phosphorylation, methylation, acetylation, sumoylation, etc Lecture 30 “Negative Control of Gene Expression in Bacteria” PPT Review 1.) What category of macromolecules would lactose fall under? Based on the double ring structure of lactose, how would it further be classified under this macromolecule family? a. Carbohydrate/Sugar b. Disaccharide 2.) What is the function of the proteins Galactoside permease and β-Galactosidase? a. Galactoside permease is a membrane protein that permits transport of lactose into the cell. Β-galactosidase cleaves lactose yielding 1 glucose and 1 galactose molecule. 3.) What is an operon? a. A region of prokaryotic DNA that codes for a series of functionally related genes and is transcribed from a single promoter into one mRNA 4.) What are the genes encoded by the lac operon promoter? Is lacI transcribed from the same lac operon promoter? Which genes express β-Galactosidase and Galactoside permease? a. lacZ, lacY, and lacA b. No lacI has its own separate promoter c. lacZ codes for β-Galactosidase and lacY codes for Galactoside permease 5.) What is an Operator in terms of prokaryotic operons? Based on its function, what would the operator be classified as? a. An operator is a binding site for a repressor protein, located near the start of an operon b. Operator is a regulatory sequence 6.) What is the function of the protein expressed from LacI? When it interacts, what part does it interact with on its target and what structure of the protein allows this? a. LacI codes for a protein (Lac repressor) that binds to the operator of the lac operon and inhibits transcription of the lac operon genes b. The Lac repressor binds DNA via a helix-turn-helix that interacts with the major groove of DNA 7.) The protein coded by the LacI gene can be altered in a way that changes its function. What is the name of this change and what induces the change to this protein? a. Allosteric change in the Lac repressor upon binding lactose will decrease its ability to bind to DNA 8.) Is the lac operon an example of positive or negative control? Based on the mechanism, explain why. How can this type of control then be suppressed? (Explain the difference between lactose being present and absent) a. Negative control: i. If lactose is absent --> the repressor is present and binds to DNA which blocks transcription because RNA Pol II is blocked from binding to the DNA (exerting negative control over the lacZ and lacY gene transcription) ii. If lactose is present--> Lactose binds to the repressor which causes the repressor to release the DNA (due to a conformational change) and therefore allow transcription to occur—because RNA Pol can now bind to the DNA 9.) How does glucose affect the transcription of the lac operon? Why? What is it interacting with to cause the effect? a. Transcription of the lac operon is drastically reduced when glucose is present in the environment. b. Glucose is the preferred carbon source in E. coli. c. It prevents transcription of the lac operon by inhibiting the lactose transport activity of galactoside permease (through a mechanism you do not need to know) Lecture 31 “Positive Control of Gene Expression in Bacteria” PPT Review 1.) What are all the components of the DNA comprising the ara operon? a. araB, araA, araD genes for enzymes required for arabinose metabolism b. araBAD promoter c. Initiator sequence d. araC gene e. araC operator f. araC promoter 2.) How does the araC act as a regulatory protein in the presence and absence of arabinose? a. When bound to arabinose – transcriptional activator for araBAD operon b. When not bound to arabinose – transcriptional repressor for araBAD operon and araC gene 3.) Slide 9 question: “The bacteria glow in response to a molecule that regulates expression of genes involved in light-producing chemical reactions. The regulator controls production of the genes’ mRNA. Therefore, the light-producing genes are under ____ “ a. Transcriptional Control 4.) Explain the LuxR regulation occurring on Slide 11. a. Expression of LuxI leads to inducer molecules recruited into the cell b. LuxR binds to an inducer molecule c. Interaction of LuxR-inducer activates transcription of additional LuxI and the C,D,A,B,E light producing genes, while also inhibiting transcription of additional LuxR 5.) Slide 12 question: LuxR is allosterically regulated by the signaling inducer molecule secreted by V. fischeri. What does it mean that LuxR is allosterically regulated? a. LuxR activity is regulated by a change in shape (conformational change) 6.) What is a regulon? What is the benefit of regulon for bacterial survival? How are they different from operons? a. A large set of genes or operons in bacteria that contain the same regulatory sequences and are controlled by a single type of regulatory molecule b. The regulon can possess a variety of operons/genes that can respond to environmental cues and respond to changing environment i. such as shortage of nutrients, sudden changes in temperature, exposure to radiation, or shifts in habitat c. Regulons can be scattered all across the genome but their transcription is still controlled by the same regulatory protein, whereas the genes in an operon are located adjacent to each other i. Regulons can also contain operons within them 7.) How does Vibrio cholera respond to quorum sensing in the human gut? a. Activates the ToxR regulon which ultimately leads to the cholera toxin 8.) Slide 16 question: “Why might quorum sensing be beneficial to pathogenic bacteria?” a. Pathogenic bacteria may benefit from mounting a full frontal assault, releasing a toxin only when there are enough cells to produce sufficient toxin to alter the metabolism of the infected host Lecture 32 “Chromatin Remodeling and Regulation during Transcription Initiation” PPT Review 1.) What are the different levels at which control of eukaryotic genes can occur? a. Can occur at: i. Transcription Initiation ii. Post-transcription iii. Translation iv. Post-Translation 2.) What problem with the eukaryotic genome did the discovery of chromatin solve? a. Eukaryotic genome contains 2 meters of DNA but is confined within a 10 micrometer space 3.) What are histones? What are nucleosomes? a. Histones = one of several positively charged proteins associated with DNA in the chromatin of eukaryotic cells b. Nucleosomes = A repeating bead-like unit of eukaryotic chromatin, consisting of about 200 nucleotides of DNA wrapped twice around eight histone proteins 4.) What is the gene promoter? In order for RNA Polymerase to gain access to the promoter, what state must chromatin be in? a. A sequence of nucleotides in the DNA that binds to basal transcription factors which then recruit RNA Polymerase to the sequence to initiate transcription b. Chromatin must be decondensed 5.) What are the 3 ways (from lecture) that chromatin can be altered? 1. DNA methylation 2. By enzymes that catalyze the acetylation (or methylation) of histones 3. ATP-dependent chromatin-remodeling complexes 6.) What is acetylation? How does acetylation of histones affect chromatin structure? What enzymes catalyze this? What enzymes reverse the acetylation? a. Acetylation = addition of an acetyl group (-COCH3) to a molecule b. Acetylation of histones neutralizes the positive charge of histones (making it neutral) therefore its interaction with DNA (net negatively charged) weakens and causes the chromatin to decondense—whereas the removal of the acetyl groups will cause it to become condensed again c. Histone acetyltransferases (HATs) acetylate histones d. Histone Deacetylases (HDACs) deacetylates chromatin 7.) What is a promoter proximal element? How does it differ from the gene’s promoter? What is the benefit of the promoter proximal elements for gene expression? a. PPE = A regulatory sequence in DNA that is close to a promoter and can bind regulatory transcription factors b. The gene’s promoter interacts with basal transcription factors and is the site that RNA Pol II is recruited to during transcription initiation. Whereas the PPE interacts with regulatory transcription factors that then assist in recruiting RNA Pol II c. PPEs have sequences that are unique to specific sets of genes and therefore express specific genes and not others (so it’s another way to control expression) 8.) What are the steps for transcription initiation in eukaryotes? 1. Transcriptional activators bind to DNA and recruit chromatin-remodeling complexes and HATs 2. A portion of chromatin is opened which exposes the promoter, PPE, and enhancers 3. Other activators bind to the newly exposed enhancers and PPEs. Basal transcription factors bind to the promoter and recruit RNA Pol II 4. Mediator complex connects the activators and basal transcription factors that are bound to DNA 5. RNA Pol II begins transcription at start site 9.) What is a transcriptional enhancer? How enhancers in the DNA work from such far distances? a. A regulatory sequence in eukaryotic DNA that may be located far from the gene it controls or within introns of the gene. Binding of proteins to enhancer promotes transcription of specific genes b. Protein-protein interactions allowing the enhancer to form a bridge to RNA Pol II bound for promoter. Lecture 33 “Post-transcriptional Control & Linking Cancer to Defects in Gene Regulation” PPT Review 1.) Is the final mature mRNA transcript composed of exons or introns? What occurs during splicing of primary mRNAs? a. The mature mRNA is made up of exons b. Splicing is the process by which introns are removed from the primary RNA transcripts and the remaining exons are ligated together 2.) What is alternative splicing? How is alternative splicing controlled? a. The process by which selected exons are removed from the primary transcript along with the introns. This results in different combinations of exons in the mature mRNA and therefore different proteins as a result. b. Alternative splicing is controlled by cell-type-specific proteins that bind to RNAs in the nucleus and interact with spliceosomes to influence which sequences are used for splicing 3.) What are microRNAs (miRNAs)? What post-transcriptional process are they involved in? What protein complex do miRNAs interact with during this process? a. Small, single-stranded RNA processed from a longer premiRNA transcript which have the ability to bind complementary to mRNA which leads to degradation of the mRNA or inhibition of its translation b. RNA Interference c. RNA-inducing silencing complex (RISC) 4.) What are the steps in the post-transcriptional regulatory process that miRNAs are involved in? a. Transcription of miRNA gene b. Precursor miRNA formed by initial processing of transcript in nucleus c. Double-stranded miRNA formed when enzyme in cytoplasm trims the RNA hairpin into a short dsRNA d. Mature miRNA formed when ds-miRNA binds to RISC protein complex and one strand is degraded e. miRNA, held by RISC, binds to complementary sequence on target mRNA f. RISC either cuts mRNA or prevents the mRNA from being translated 5.) Why is RNAi also referred to as gene knockdown? a. This method reduces gene expression but not 100% removal of gene expression 6.) What is a tumor suppressor gene? Why are they associated with cancer? What example of a tumor suppressor did we talk about in lecture? a. A gene that codes for a protein that prevents cell division, such as when the cell has DNA damage b. Mutation in a tumor suppressor can result in unregulated, accelerated cell division which may allow for damaged DNA to be replicated c. P53 7.) What effect might too much HDAC have on 1.) expression of p53 and 2.) the cell cycle? a. Expression of p53 would be decreased b. ^^therefore decreasing p53 expression would cause the cell cycle to be less regulated and cycle faster due to the decrease in checkpoints for DNA repair (that are regulated by p53) 8.) What is a proto-oncogene? How does it differ from an oncogene? a. Any gene that encourages cell-division in a regulated manner, typically by triggering specific phases in the cell cycle b. An oncogene is a mutation in the proto-oncogene that causes expression of a protein product that stimulates cell division at all times and thus promote cancer development 9.) What effect might too much HAT have on 1.) expression of a proto-oncogene and 2.) the cell cycle? a. Chromatin would be more decondensed, possibly exposing proto-oncogene and therefore may upregulate the expression of the proto-oncogene b. ^^Having upregulated proto-oncogene may accelerate the cell cycle 10.) How can translation be slowed or stopped (ex. From class)? What type of scenario could this occur in? Why might it be useful to the cell to stop most protein synthesis in these conditions? a. By phosphorylation of a translation initiation factor b. In response to a sudden increase in temperature or viral infection c. If viral infection, inhibiting translation of viral proteins (since viruses use our cell machinery to replicate its genome) would be beneficial to prevent viral outbreak in body Lecture 35 “Principles of Development” Review 1.) What are the five essential developmental processes we discussed? a. Cell Proliferation, Cell-cell interactions, Cell differentiation, Cell movement and expansion, and Programmed cell death i. Page in the Dev text chapter has a table showing and briefly explaining each 2.) What were the steps in cloning? 1. Obtain mammary cell from one donor and egg cell from another donor 2. Culture mammary cells and remove nucleus from egg cell 3. Fuse cells 4. Nucleus of mammary cell enters egg cell 5. Grow Embryo 6. Implant embryo in surrogate mother (review cloning movie) 3.) With the cloning procedure that we discussed, would the offspring have identical genetic information as the embryo donor or the mammary cell donor or neither? a. Identical to mammary-cell donor 4.) Be familiar with the anatomical terminology for this test: a. Anterior, Ventral, Dorsal, Posterior 5.) Eukaryotic cells control gene expression at several different levels: chromatin remodeling and modification, transcriptional regulation, alternative splicing of mRNAs, selective destruction of mRNAs, translation rate, and activation and deactivation of proteins after they’re translated. All occur during development, but which is the most important in differentiation? Why? a. Transcriptional regulation/control—A specific cell type will transcribe specialized genes required specifically for their cell type and not specialized genes for other types of cells (instead of transcribing all specialized genes somewhat haphazardly which would then require that the resulting nonessential mRNAs/proteins be inhibited or degraded) 6.) How was pattern formation defined in lecture? a. The establishment of the spatial organization of an embryo 7.) What is a morphogen? When we discussed embryos expressing the bicoid protein, what determined the anterior and posterior axes? Bicoid’s interaction with DNA classifies it as what type of protein? a. A morphogen is “a molecule that exists in a concentration gradient and provides spatial information to embryonic cells” (text) b. A concentration gradient of the bcd (bicoid) mRNA (and eventually protein)—high [bcd] led to ANT segment c. Bicoid is a regulatory transcription factor 8.) Aside from “maternal-effect genes” like bicoid that control formation of large segments like the ANT/POST axes, what are the other 3 types of segmentation genes? What segments are being defined by each? a. Gap genes—defines broad regions that often span several segments b. Pair-rule genes—define individual segments c. Segment polarity genes—delineate regions within the individual segments 9.) In what order would each of the segmentation genes be expressed? (The regulatory cascade of transcription factors) a. Maternal-effect genes (morphogen) Gap genes Pair-rule genes Segment Polarity genes 10.) What are homeotic genes? What is the homeotic gene discussed in class? a. Genes that trigger the development of the structures appropriate to each type of segment b. Hox gene discussed in class 11.) What is genetic equivalence? What is differential gene expression? How are cell types influenced by differential gene expression? a. Genetic equivalence = Having all different cell types of a multicellular individual possess the same genome b. Differential Gene Expression = Expression of different sets of genes in cells with the same genome. c. This is responsible for creating different cell types 12.) What do HOX genes encode? What is the sequence that distinguishes hox genes? What type of external factors disturb the regulation of HOX genes? a. Hox genes encode transcription factors b. The sequence within hox genes is called a homeobox that encodes a DNA-binding domain (homeodomain) c. Environmental factors (review the example with Vitamin A) Lecture 36 “Genes, Development, and Evolution” PPT Review 1.) What is the focus of evo-devo biologists? What example was discussed in class? a. Biologists that study how changes in developmentally important genes lead to the evolution of new phenotypes b. The way in which snakes “lost” their limbs through changes in expression of homeotic genes 2.) Chick Embryo slide: What gene(s) must be expressed for the forelimb to form? What gene(s) must be expressed for the ribs to form? Using this, why are there no forelimbs in snakes? a. Forelimb—Hoxc6 only b. Ribs—Hoxc6 and Hoxc8 c. They express both Hoxc6 and Hoxc8 at all locations where the forelimbs should develop and for forelimb development only Hoxc6 can be expressed 3.) Snake example: what would cause them to “lose” their hindlimbs? When this pathway is functioning “normally”, what is its immediate function? a. Defect in sonic hedgehog (SHH) b. Determines segment polarity (ANT vs POST) 4.) Through what type of cell communication pathway does SHH function? What are PTCH and SMO? What is GLI? a. SHH functions through a signal transduction pathway b. PTCH and SMO are receptors c. GLI is a transcription factor (keep in mind it is therefore a nuclear protein—has NLS + associated importin) 5.) Based on the diagram given, propose the basic steps that occur in the SHH pathway. a. SHH binds to PTCH b. Interaction between SHH and PTCH releases the inhibition of SMO by PTCH c. GLI then becomes activated d. GLI enter nucleus and binds to DNA e. Change in gene expression 6.) What is a tool-kit gene? For the whale example, what caused the loss of hind limbs? a. A set of key developmental genes that establishes the body plan of animals and plants b. Defect in SHH was cause for loss of hind limbs 7.) When sperm and egg are still gametes, are these cells diploid or haploid? What is the process of these two fusing together? When they are fused together, is the product diploid or haploid? a. Both are haploid after gametogenesis b. Fertilization is the process of the two fusing c. After fertilization, the fertilized egg is diploid 8.) What occurs during the cleavage phase of development? What occurs during gastrulation? a. Cleavage is a series of rapid mitotic cell divisions, with little cell growth, that produces successively smaller cells (blastomeres) and transforms a zygote into a multicellular blastula (text) b. Gastrulation is the process of coordinated cell movement, including the moving of some cells from the outer surface of the embryo to the interior, resulting in the formation of three germ layers and the axes of the embryo (text) i. The movement of cells to generate distinct developmental regions within the embryo 9.) Why are sea urchins a model system for studying fertilization? a. Their development is initiated by EXTERNAL fertilization 10.) What is organogenesis? What are the steps for developing a complete neural tube? What is the purpose of the neural tube? a. The process by which cells become assembled into recognizable tissues and organs b. First notochord forms, then notochord signals the ectoderm to fold. Once it does, this results in the completion of the neural tube c. This is the site for the development of brain and spinal cord 11.) What is a somite? What development can result from somites? a. A block of mesoderm that occurs in pairs along both sides of the developing neural tube in a vertebrate embryo. b. Gives rise to muscle, vertebrae, ribs, and the dermis of the skin 12.) What occurs when a cell becomes determined? What influences how cells in somites become determined? a. The cell becomes committed to a particular differentiated fate—it differentiates into only a particular cell type b. Influenced by positioning in somites 13.) What caused the cells in a certain part of somites to become committed to produce muscle? a. Certain somite cells (myoblasts) produce an mRNA for a regulatory protein that commits them to differentiate into muscle 14.) What does MyoD stand for? What does the MyoD gene encode? What does the MyoD protein do? a. MyoD stands for myoblast determination. b. The MyoD gene encodes a regulatory transcription factor (MyoD) c. MyoD binds to enhancer elements located upstream of muscle-specific genes. Lecture 37 “Molecular Revolution: Molecular Biology and Genomics” PPT Review 1.) What are restriction endonucleases? What is DNA ligase? How are they both involved in recombinant DNA technology? a. Bacterial enzymes that cut DNA at specific locations b. Enzyme that joins DNA segments together c. The combination of the two allows isolation of a specific DNA fragment and then introduction of that fragment into different DNA regions or different hosts 2.) What is complementary DNA (cDNA)? Does cDNA contain introns? Are regulatory DNA sequences present in cDNA? What part of the gene is present in cDNA? a. DNA produced using an RNA transcript as a template and reverse transcriptase b. No introns in cDNA c. No regulatory sequences in cDNA d. Coding region 3.) What is a cDNA library? How are the cDNAs stored? What are the steps in creating a cDNA library? a. A set of cDNAs from a particular cell type of stage of development. b. Each cDNA is carried by a plasmid or other cloning vector and can be separated from other cDNAs c. Isolate mRNAs --> Synthesize cDNA using reverse transcriptase --> Make cDNA doublestranded --> Make recombinant plasmid --> Transformation into E. coli 4.) What is a plasmid? How can they be used to produce a high concentration of a gene of interest? a. Plasmid is a small, circular extrachromosomal DNA molecule capable of autonomous replication in a cell b. A gene of interest is isolated from a DNA source and then inserted into a bacterial plasmid using restriction endonucleases and DNA ligase. The bacterial cell can then be given optimal conditions for growth. The bacteria will then replicate numerous times in a given time frame, each time producing a copy of our gene of interest which can then be isolated and used for other purposes. 5.) What does dideoxy sequencing reveal? When a dideoxynucleotide triphosphate (ddNTP) binds to DNA, what happens? Why does this happen? a. Determines the exact nucleotide sequencing of DNA b. ddNTPs terminate DNA replication c. ddNTPs lack a hydroxyl group at the 3’ carbon. Therefore, no hydroxyl is available on the 3’ carbon to link to the 5’ carbon of the incoming dNTP monomer which causes termination of synthesis. 6.) What goes into the reaction mixture of dideoxy sequencing? What are the steps in dideoxy sequencing? How are the ddNTPs identified after forming strands? a. Numerous dNTPs, few ddNTPs, template DNA, primer for the target sequencing, DNA polymerase b. Incubate reaction mixture --> DNA synthesis then occurs and strands will be labeled with ddNTPs --> Collect DNA strands that are produced --> Separate fragments via electrophoresis --> Read output (done by automated sequencing machine) c. The ddNTPs are fluorescently labeled and can then be detected 7.) What is genomics? What is bioinformatics? a. Genomics = The effort to sequence, interpret, and compare whole genomes b. Bioinformatics = The effort to manage, analyze, and interpret information, particularly DNA sequences 8.) What do functional genomics and proteomics allow us to analyze? a. Allows us to analyze how different genes and gene products within an organism interact 9.) What is the benefit of DNA microarrays? a. Allows researchers to measure the expression of every gene in the genome simultaneously 10.) What are the steps in using a DNA microarray? 1. Isolate mRNAs from control cells and treatment cells 2. Prepare ss-cDNA using reverse transcriptase 3. Label cDNA using fluorescent tags 4. Hybridize a microarray—the labeled cDNAs will bind complementary DNA probe sequences on the slide 5. Shine laser light to induce fluorescence 6. Observe which genes are being transcribed (and the rate of transcription) 11.) What are transposable elements? What is lateral gene transfer? a. TE = DNA sequences that have the ability to integrate into the genome at a new site within their cell of origin b. Lateral gene transfer = transfer of DNA between two different species