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Exam 3 SQ Key Chapter 16: How Genes Work Lecture 21 “How Genes Work: An Overview of the Central Dogma” 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) Chapter 17: Transcription, RNA Processing, and Translation Lecture 22 “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 23 “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. Ribsomes 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 cytoplasm. This allows for more control of when the resulting mRNA will be expressed in eukaryotes. 2.) What is the function of a polyribosome? a. Allows many copies of a protein to be produced from a single mRNA 3.) 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 4.) 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 5.) What is the term used to describe a tRNA molecule covalently linked to an AA? a. Aminoacyl tRNA 6.) 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 7.) 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 8.) 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 9.) 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 3. Large subunit then binds 4. Translation can now occur 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.) What is the function of a molecular chaperone? a. Chaperones facilitate correct protein folding and assist in speeding up correct protein folding 15.) Why might a protein need to undergo a post-translational modification? What are the 4 posttranslational modifications that were discussed in lecture? a. PTMs often activate some proteins or target them to specific locations b. Phosphorylation, methylation, acetylation, sumoylation Chapter 18: Control of Gene Expression in Bacteria Lecture 24 “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? a. Negative control: i. If lactose is absent, the repressor is present and binds to DNA which blocks transcription (exerting negative control over the lacZ and lacY gene transcription) b. If lactose is present--> Lactose binds to the repressor which causes the repressor to release the DNA and therefore allow transcription to occur. 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) 10.) 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 11.) What are the basic principles of gene regulation from lecture? 1. Gene expression is regulated by physical contact between regulatory proteins and regulatory sites within the DNA 2. RNA Polymerase works alone with a certain frequency 3. Transcriptional activators increase the frequency 4. Transcriptional repressors decrease the frequency 5. Repressors and activators are DNA-binding proteins Chapter 19: Control of Gene Expression in Eukaryotes Lecture 25 “Control of Gene Expression in Eukaryotes: Part 1” 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 4.) 5.) 6.) 7.) 8.) 9.) b. Nucleosomes = A repeating bead-like unit of eukaryotic chromatin, consisting of about 200 nucleotides of DNA wrapped twice around eight histone proteins 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 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 What is acetylation? How can does acetylation of histones affect chromatin structure? What enzymes catalyze this? a. Acetylation = addition of an acetyl group (-COCH3) to a molecule b. Acetylation of histones causes chromatin to become decondensed, whereas the removal of the acetyl groups will cause it to become condensed again c. Histone acetyltransferases (HATs) acetylate histones and Histone Deacetylases (HDACs) deacetylate 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) 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 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 26 “Control of Gene Expression in Eukaryotes: Part 2” 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 Chapter 20: Analyzing and Engineering Genes Lecture 27 “Analyzing and Engineering Genes” 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? 5.) 6.) 7.) 8.) 9.) 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. What is a transgenic animal? a. An animal whose genome contains DNA introduced from another individual, often from a different species 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. 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 What are three types of genetic testing? a. Carrier testing, prenatal testing, and adult testing What does a genetic map reveal and how is it revealed? What does a physical map reveal? a. Shows the relative positions of genes on the same chromosomes determined by analyzing the frequency of recombination between pairs of genes b. Physical map shows the absolute position of a gene—in numbers of base pairs—along a chromosome Chapter 21: Genomics and Beyond Lecture 28 “Genomics & Beyond” PPT Review 1.) 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 2.) What are the different applications of bioinformatics discussed in lecture? a. Locate and align sequences 3.) 4.) 5.) 6.) 7.) 8.) b. Assemble consensus sequences c. Analyze properties of proteins d. Analyze sequence patterns to locate restriction sites, promoters, enhancers, etc. e. Phylogenetic analysis 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 What is the benefit of DNA microarrays? a. Allows researchers to measure the expression of every gene in the genome simultaneously 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 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 What is the CRISPR/Cas system and where does it originate? What does CRISPR stand for? Cas? a. Bacterial system that recognizes and removes invading viral DNA b. CRISPR = Clustered Regularly Interspaces Short Palindromic Repeats c. Cas = CRISPR Associated genes How is the CRISPR-Cas system being used in gene editing? a. The system can be engineered to cut any DNA sequence by changing the guide RNA to match the target. Therefore, this can be used in any organism to possibly remove one gene and replace it with a desired gene Chapter 22: Principles of Development Lecture 29 “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 406 in the text 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) p. 409 in text 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? 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 30 “Evolutionary Developmental Biology” Review of Material in Chapter 22: 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 by itself 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.) Still considering the snake example, what would cause them to “lose” their hindlimbs? When this 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 Chapter 23: An Introduction to Animal Development Lecture 30 “Evolutionary Developmental Biology” Review of Material in Chapter 23: 1.) 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 2.) 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 (PPT Lec 31) 3.) Why are sea urchins a model system for studying fertilization? a. Their development is initiated by EXTERNAL fertilization 4.) What type of molecule is the jelly layer of eggs composed of? What are cortical granules? a. Composed of glycoproteins b. Cortical granules are small vesicles filled with enzymes 5.) What is an acrosome? What do acrosomes contain that assists in fertilization? What allows for movement of sperm cells? a. A caplike structure, located on the head of a sperm cell. b. They contain enzymes capable of dissolving the outer coverings of an egg c. Flagella—flagellar movement/propulsion 6.) What are the steps of fertilization (according to PPT)? a. Receptors on sperm come into contact with egg jelly b. Acrosome reaction—contents are released. Enzymes digest a path through the egg jelly c. The actin pushes the front of the sperm outward (Acrosomal process) exposing the species-specific bindin proteins d. Bindin binds with a species-specific receptor on the surface of the egg Lecture 31 “Comparative Development” Review: 1.) What is polyspermy? What affect does it have with genetic material? a. Polyspermy is the fertilization of an egg by multiple sperm b. It would result in the zygote having more than two copies of each chromosome 2.) How is polyspermy prevented in sea urchins? a. Once a sperm enters the egg, the egg releases proteases that digest exterior-facing portion of receptor for other sperm (more to the process in text p. 421-422) 3.) What are cytoplasmic determinants? What stage of development is directly influenced by these? a. A regulatory transcription factor or signaling molecule that is distributed unevenly in the cytoplasm of the egg and that directs early pattern formation in an embryo b. Cleavage 4.) How does the production of new mRNA differ between frog and mammals during development? a. In frog, new mRNAs aren’t made until after the 12th cleavage. In mammals, mRNAs are transcribed from the zygotic genome at the 2-cell stage. 5.) In humans, does cleave occur before or after implantation into the uterus? a. Before 6.) 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 7.) 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 8.) 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 9.) 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 10.) 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. Chapter 24: An Introduction to Plant Development (24.3) Lecture 32 “Comparative Development Part II” PPT + 24.3 Review: 1.) As a flower develops, their floral meristem produces which four kinds of organs? a. Sepals, Petals, Stamens, and Carpels 2.) What is MADS-box? a. A DNA sequence that codes for a DNA-binding motif in proteins that is present in floral organ identity genes in plants 3.) What are the ways in which MADS-box genes in plants and Hox genes in animals are similar? a. Both encode DNA-binding transcription factors b. They are both part of regulatory cascades that lead to the specification of structures i. Hox regulate expression of genes responsible for specific parts of body ii. MADS-box regulate the expression of genes responsible for forming flowers 4.) 5.) 6.) 7.) 8.) 9.) c. Both sets of genes tell cells where they are in the body and can produce homeotic mutants if they do not work properly What type of plasmid do T-DNA originate from? Why are these plasmids, but more so the bacteria that possesses them, used for introducing recombinant genes into plants? a. Tumor inducing (Ti) Plasmid b. Agrobacterium infects plant tissue. The Ti Plasmids encode proteins that bind to the cell walls of plant cells and other proteins that allow the T-DNA to enter the cellnucleus Does T-DNA remain extra-chromosomal or does it integrate into the hosts chromosomes? a. Integrates into the host-cell chromosomes In order to replace the tumor inducing gene and replace it with another gene of interest, what are the two enzymes that would allow the removal of the Ti gene and integration of the new gene? a. Restriction endonucleases to remove Ti and Ligases to ligate new gene into plasmid What were the steps taken to integrate Edward Kac’s Immunoglobulin (Ig) light chain into a plasmid vector? 1. Obtain sample of Kac’s blood 2. Extract coding region of Ig light chain 3. Remove Ti gene of Agrobacterium plasmid with endonucleases 4. Add Kac’s Ig gene into Ti plasmid with promoter Does a promoter need to be added to the T-DNA containing our gene of interest? Explain why. a. Yes because the coding region for the gene of interest contains no regulatory regions i. Similar reasoning to the addition of cDNA to plasmid vectors What other component must be included in the T-DNA plasmid in order for the growth of only recombinant plasmids (that contain our gene of interest)? a. Gene for antibiotic resistance—therefore, the cells that possess these plasmids can be grown on a media with the antibiotic that the plasmid contains antibiotic resistance to—yielding only growth of cells that have our recombinant DNA incorporated into them