LS7A final learning objectives
Describe the role of histone proteins in DNA packaging, nucleosome formation, and chromatin structure
-In eukaryotic cells, DNA is packaged as one molecule per chromosome. -At the base level, DNA is wrapped twice around a group of histone proteins (which are highly alkaline proteins) called a nucleosome. -These nucleosomes become even further coiled into chromatin fibers which are 300 nm in diameter. -As the chromosomes condense in preparation for cell division, they become short and thicker until they become the 1400 nm condensed chromatin.
Label a diagram of the lac operon and state the function of each component
-Lacl promoter (P)- ensures whether lacl gene is expressed -Lacl gene (I) - produces repressor protein; regulates lacO gene -Lac(O) - operator that repressor protein binds to -LacZ - gene (coding sequence) for the enzyme β-galactosidase, which cleaves the lactose molecule into its glucose and galactose constituents. A functional, nonmutant form of the -lacZ gene is denoted lacZ+ (hi cathy how are u -- doing good buddy?) -LacY - the gene (coding sequence) for the protein lactose permease, which transports lactose from the external medium into the cell -CRP-CAMP binding site - region of DNA adjacent to promoter Absence of glucose creates an increase in CAMP that activates CAP and allows RNA polymerase to associate with promoter Presence of glucose decreases CAMP....
Describe how the lac repressor and CRP regulate expression of the lac operon
-Repressor prevents transcription from taking place -CRP stimulates transcription
Discuss how histones are chemically modified to increase or decrease the expression of a gene
Acetylation: occurs at the histone tails and adds an acetyl group to them, which allows DNA binding proteins to interact with exposed sites to activate gene transcription and downstream cellular functions Methylation: adds a methyl group to a cytosine which can recruit proteins that affect changes in chromatin structure, histone modification, and nucleosome positioning that restrict access of transcription factors to gene promoters. Occurs near CPG islands (clusters of adjacent CG nucleotides located near or on the promoter gene) Histones have histone tails made from strings of amino acids. These can be methylated or acetylated, each having a different effect on the histone. Acetyl groups tend to make chromatin looser, while methyl groups condense it. The combined result of these modifications is called the histone code.
Describe how alternative splicing allows for multiple gene products to be produced from the same gene
Alternative splicing is a process in which the same primary transcript can be spliced different ways to yield different proteins in the process What one splicing machinery can see as an exon in some primary transcripts, it recognizes as part of an intron in another. Spliceosomes in different tissues can recognize different introns and exons and create new mRNA from the same gene, therefore coding for many more proteins than we have genes.
Evaluate data to compare and contrast mutation rates across organisms
Based on data, mutation is rare event in individual nucleotides in humans but common across the entire genome - highest mutation rate = viruses (use RNA as genetic material and RNA polymerase does not have proofreading activity) - lowest = human
Predict whether gene expression of an operon will occur given specific environmental conditions
CAMP = activates it Glucose = does not Lactose = does not high glucose = low cAMP = CRP does not bind to DNA = low transcription low glucose = high cAMP = CRP-cAMP binds to DNA = high transcription
Interpret DNA fingerprinting data (VNTR/STR analysis) to identify an individual or characterize genetic diversity among different organisms
Can identify individuals using samples of DNA by examining similar tandem repeats Genetic diversity is based on the existence of multiple alleles (different numbers of repeats)
Explain how chromatin packaging affects gene expression in eukaryotes
Chromatin is packaged into nucleosomes, which are just chunks of DNA wrapped around histone proteins. When it is tightly wound, it is difficult for transcription factors to bind to promoters in the gene. Therefore, packaging (the tighter it is) represses gene expression. Euchromatin is the less condensed and more transcriptionally active form. Heterochromatin is more condensed and less transcriptionally active. need chromatin remodeling to move nucleosomes to access DNA sequence
Explain why the appearance of DNA changes from interphase to mitosis
Chromosomes condense
Explain the function of components of the replication machinery
DNA polymerase - enzyme that is a critical component of a large protein complex that carries DNA replication Primase - short stretch of RNA at the beginning of each new DNA strand that serves as a starter for photosynthesis Helicase - protein that unwinds the parental double helix of the replication fork Topoisomerase - an enzyme that breaks the DNA double helix, rotates the ends, and seals the break Single-stranded binding protein - a protein that binds single stranded nucleic acids Ligase - an enzyme that uses the energy in ATP to close a nick in a DNA strand, joining the 3' hydroxyl of one end to the 5' phosphate of the other end
Predict how drugs or mutations that alter the process of mitosis will affect cell growth
Drugs or mutations that alter mitosis could inhibit cytokinesis cause cells with multiple nucleotides see clicker about microtubules and mitosis cell-cycle checkpoint
Predict how the presence of drugs or mutations that alter cell cycle regulation will affect cell growth and division
Drugs or mutations that increase oncogenes or decrease tumor suppressors will increase cell growth and division = cancer Drugs or mutations that decrease oncogenes or increase tumor suppressors will decrease cell growth and division = not cancer Drugs or mutations that inhibit regulation at checkpoints could lead to division of cells that are unprepared or not completely formed loss of function in tumor suppressor (brake) gain of function in proto-oncogene (gas)
Relate the quantity of DNA found in a cell to the cell's progression through the cell cycle
G1 - cell prepares for DNA replication; have one genome worth of DNA S - cells actively replicating DNA; 1-2 genome worth of DNA G2 - after DNA replication; two genome worth of DNA G1 - original DNA S - original DNA, preparing to double G2 - double Mitosis - original amount Cyto - original amount
Relate the terms "homologous chromosome" and "sister chromatid" to the process of mitosis and the segregation of genetic information
Homologous chromosomes are chromosomes that encode the same genes. You have 23 pairs, 46 total. One comes from mom, the other from dad. Sister chromatids only appear after replication. They are a copy of each chromosome, connected at the centromere. Homologous chromosomes will each have a sister chromatid, and these will be pulled apart at anaphase so that each daughter cell still has 46 total chromosomes and 23 pairs of homologous chromosomes. homologous chromosomes - have same genetic info sister chromatids - replicated pair of single chromosome each cell receives a separated sister chromatid (now chromosome) to have same amount of DNA as original
Relate DNA repair processes (i.e., BRCA1) to the regulation of the cell cycle
If DNA is deformed, cell cycle will stop at G2 checkpoint and DNA repair processes will ensure that full set of chromosomes replicated before cell can enter mitosis and cytokinesis G2 checkpoint before mitosis checks if all DNA damage is repaired ex: BRCA1 fixes double strand breaks
Predict changes in PCR results based on alterations of required components
If DNA polymerase is altered during the process of denaturation, will not be effective Use Taq polymerase from bacteria instead that can withstand high temperatures - no primers = no new DNA copies - no Taq polymerase/no primers/no dNTPS = no DNA
Discuss the possible consequences that could arise if DNA is not properly repaired
If a DNA is not properly repaired, it can cause diseases and/or genetic variations
Discuss the possible consequences that could arise if DNA is not properly repaired
If not properly repaired, mutation will get transmitted to future generations (in a germline mutation as in egg and sperm cells) or affect only the individuals (such as somatic mutations)
Identify changes in gene expression based on the haplotypes found in the cell (i.e. using partial diploids)
If repressor protein works on either cell, in the absence of an inducer, gene will not be expressed If the operon is constitutive on any operon, then it will still be expressed Operons cannot move but repressors can partial diploid cells contain 2 copies of lac operon and 2 copies of the lacI gene
Analyze PCR results as they relate to presence or absence of a region in the genome of multiple organisms
If shows up on PCR gel, contains region of genome If does not show up on PCR gel, does not contain region of genome
Evaluate how changing components of the replication machinery would alter the process of replication
If you take out dna of the replication machine, it doesn't work!!!\ If you take out DNA polymerase, you cannot add nucleotides that will make the new strand If you take out primase, DNA polymerase cannot add nucleotides bc it can only add nucleotides to an existing chain If you take out helicase, you cannot unwind the double helix and create the replication fork If you take out topoisomerase, you cannot break the double helix, rotate the ends, and seal the break If you take out the single-stranded binding protein, you cannot bind the single stranded nucleic acids If you take out ligase, you cannot join the okazaki fragments together
Discuss the role of Xist in regulating gene expression at the chromosome level and why this is important for regulating gene dosage
In X-activation, a X-inactivation RNA transcript becomes transcribed at an increased level and binds to the x chromosome inactivation center in an X chromosome. As it accumulates, the coating spreads outwards from the XIC until the entire chromosome is coated with Xist RNA. The presence of Xist RNA recruits factors that promotes DNA methylation, histone modification, and other changes associated with transcriptional repression. Xist is a gene in the XIC region of an X chromosome. The Xist gene encodes a noncoding mRNA that coats the XIC region and eventually spreads over the whole chromosome. This promotes methylation, histone modification, and facilitates other transcriptional repression.
Discuss the relationship between gene number, genome size, and organismal complexity
In eukaryotes, there is no relationship between genome size and organismal complexity. The range of genome sizes is huge even among similar organisms. C-value paradox - you cannot predict genome size from the complexity of an organism (C being the amount of DNA in a reproductive cell) Genomes of diff species contain vastly differing amounts of repetitive DNA due to the evolution of transposable elements.
Outline the stages of the cell cycle
Interphase - cell makes preparations for division Interphase - time between 2 successive M phases; where cell makes many preparations for division (replicates DNA in the nucleus and increases cell size) -G1 phase - specific regulatory proteins are made and activated; these are kinases that promote the activity of enzymes that synthesize DNA -S Phase - phase of interphase in which the DNA content of the nucleus is replicated -G2 phase - both the size and protein content of the cell increase in preparation for cell division (M-phase mitosis and cytokinesis) ***G0 phase - absence of preparations for DNA synthesis; cell pause in the cycle cycle between M Phase and S Phase (may last for periods that range from days to years) Mphase - consists of two different events: mitosis (separation of chromosomes into two nuclei) and cytokinesis (division of cells itself into two separate cells)
Relate events in the process of DNA replication to stages in the cell cycle
Interphase - cell makes preparations for division Interphase - time between 2 successive M phases; where cell makes many preparations for division (replicates DNA in the nucleus and increases cell size) G1 phase - specific regulatory proteins are made and activated; these are kinases that promote the activity of enzymes that synthesize DNA S Phase - phase of interphase in which the DNA content of the nucleus is replicated G2 phase - both the size and protein content of the cell increase in preparation for cell division (M-phase mitosis and cytokinesis) ***G0 phase - absence of preparations for DNA synthesis; cell pause in the cycle cycle between M Phase and S Phase (may last for periods that range from days to years) Mphase - consists of two different events: mitosis (separation of chromosomes into two nuclei) and cytokinesis (division of cells itself into two separate cells)
Describe the stages of mitosis
Interphase - consists of G1, S, and G2 Prophase - chromosomes condense and centrosomes radiate microtubules and migrate to opposite poles Prometaphase - microtubules of the mitotic spindle attach to chromosomes Metaphase - chromosomes align in the center of the cell Anaphase - sister chromatids (which become individual chromosomes when the centromere splits) separate and travel to opposite poles Telophase and Cytokinesis - nuclear envelope reforms and chromosomes decondense.
Differentiate between the leading and lagging strand during DNA replication
Leading strand = grows as a single strand Lagging strand = shorter, okazaki fragments leading strand - synthesized continuously; same direction as overall replication lagging strand - synthesized in pieces; opposite direction of overall replication
Discuss the "end replication problem" and how it is dealt with by cells with linear chromosomes
Linear DNA molecules have ends and at each round of DNA replication, the ends become shorter and shorter This is due to the fact that the RNA primer cannot be synthesized precisely at the 3' end of a DNA strand when the last RNA primer in the lagging strand is removed Eukaryotic organisms have evolved to solve the problem of shortened ends by having each end capped with a repeating sequence called the telomere. -The telomere is slightly shortened in each round of DNA replication but shortened ends is quickly restored by an enzyme known as a telomerase, which carries an RNA molecule complementary to the telomere sequence. -As a result, the original telomerase and original sequences in the DNA is restored and conserved. -Telomere is made with telomerase which is an enzyme that adds an RNA primer.
Relate changes in cell cycle regulation and the process of cell division to the development of cancer
Many mutations in cell cycle regulation that lead to uncontrollable cell division can lead to the development of cancer. too much cell division
Describe how the polymerase chain reaction (PCR) produces many copies of a DNA molecule
Materials -Template DNA - serves as the template for replication -DNA polymerase - used to replicate the DNA -Deoxynucleoside Triphosphates (dNTPS) - Bases A,T,G, or C are needed as building blocks for synthesis of new DNA strands -Primers - two short seq of single stranded DNA that are required for the DNA polymerase to start synthesis Consists of 3 steps: -Denaturation - solution is heated to separate the DNA strands into two individual strands (heat short of boiling so DNA strands denature due to breaking of H bonds between complementary bases) -Annealing - great excess of primer molecules bind/ "anneal" to complementary sequence on the strands of the template duplex (template lowered to 50 to 65 degrees C in order to allow DNA primers to be annealed) -Extensions - at 72 degrees C, poly such as Taq is allowed to bind; solution is heated to the optimal temp for DNA polymerase and the polymerase elongates each primer with the deoxynucleoside triphosphates in a 5' to 3' direction
Interpret potential errors in PCR design based on gel results
Primers could not bind target sequence at all, could bind multiple spots on the sequence or bind to the wrong sequence altogether
Identify proteins and small molecules that regulate expression of the lac operon
Proteins - -Repressor - when lactose isn't present, the repressor binds to the operator and prevents transcription from taking place -CRP - binds to lac operon DNA; CRP binds to site near operator and stimulate transcription Small Molecules -Lactose - binds to the repressor protein, preventing it from binding to the operator and allows transcription to take place -cAMP - makes CAMP-CRP complex when bound to CRP; when they are large amounts of cAMP, -cAMP binds to CRP, so it can bind to a site near the operator and stimulate binding of RNA polymerase to transcribe lacZ and lacY.
Explain how CDK and cyclin regulate the progression of the cell cycle
Regulatory proteins known as cyclins create complexes with cyclin-dependent kinases to control progression through the cell cycle. A specific cyclin binds to its kinase and activates it , causing it to phosphorylate target proteins involved in each cycle of cell division For example, the G1/S cyclin-CDK complex activates a protein that promotes the expression of histone proteins needed for packaging the newly replicated DNA strands (and is necessary for the cell to enter the S phase)
Predict the size of a PCR product based on a given DNA template and primer pair
Size of PCR product based on primer on each strand - product will as long as space between the two primers
Outline the steps involved in creating a recombinant DNA plasmid
Start with a fragment of double-stranded DNA as the donor. The donor may be a protein-coding gene, a regulatory part of a gene, or any DNA segment of interest. A vector is also required. This carries the donor DNA in fashion that can be maintained and used by bacterial cells. Most often, the vector used is a plasmid, which has the ability to duplicate along with normal bacterial DNA. In order to make sure donor DNA can be fused with vector DNA, both pieces are cut with the same restriction enzyme so they both have the same overhangs. The overhangs on the donor fragment and the vector are complementary, allowing the ends of the donor fragment to renature with the ends of the opened vector when the two molecules are mixed. Last is transformation, where bacterial cells which have been treated to intake foreign DNA absorb the plasmid and allow it to multiply along with its own DNA.
Evaluate the effect of different chromatin modifying enzymes on gene expression
Swi/Snf is a chromatin remodeling complex that uses ATP to slide nucleosomes down a strand of DNA Histone Acetyltransferase (HATs) add acetyl groups to histone tails causing chromatin to loosen and gene expression to increase. Histone Deacetyltranferase (HDACs) removes the modifications and causes chromatin to condense. Histone Methyltransferases (HMTs) add methyl groups to histone tails, condensing chromatin. Some proteins recognize the methylation and bind to further repress transcription. HAT (histone acetyl transferase) - acetylation = increases transcription HDAC (histone de-acetylase) - deacetylation = impedes transcription DMNT (DNA methyl transferase) - methylation = impedes transcription
Determine whether two different restriction enzymes would generate compatible "sticky ends" given the recognition sequence and cut site for the enzymes
They would only generate compatible "sticky ends" if they cleave at the same site, producing the same overhangs that can then base pair with each other.
Differentiate between transcriptional activators and coactivators and explain their roles in regulating eukaryotic gene expression
Transcriptional activators are regulatory transcription factors that bind to enhancers and then recruit the rest of the necessary transcription factors and RNA polymerase. They active transcription. Activator binds to DNA directly at enhancer. Coactivator binds DNA indirectly by binding activator. Coactivators can be used on a variety of genes to increase the rate of transcription.
Define the term "variable number tandem repeat" (VNTR) as it relates to genetic variation
VNTR - a genetic difference in which the number of short repeated seq of DNA differ from one chromosome to the next
Explain the experimental processes of DNA fingerprinting using VNTR analysis
VNTR bands on protein gel correlate to number of repeats Variable number tandem repeat (VNTR): chromosomes have different numbers of repeated sequence at particular location Different numbers of repeats correspond to different alleles and different traits = genetic variation
Compare and contrast the genetic information stored in complementary DNA (cDNA) vs. genomic DNA
cDNA is just the copy of a mature RNA, therefore it only encodes for what is necessary to build a protein through translation (intron free). Genomic DNA in eukaryotes contains introns and untranslated regions. cDNA = DNA made from RNA; no introns; made by reverse transcriptase
Identify potential restriction enzyme sites based on the sequence of nucleotides
palindromic sequence (read same in both directions)
Determine the size(s) of restriction fragments produced when a linear or circular piece of DNA is cut by one or more restriction enzymes
see clicker questions -make sure if there are both sizes like 5, it only appears on the gel once
Predict the effect of mutations in gene regulatory elements on gene expression
see clicker questions: - if HAT cannot bind to histone = no acetylation = DNA cannot be read/expressed - no hydrolysis of ATP = no energy to move nucleosomes = RNA polymerase cannot get through - enhancer sequence mutated so activator protein cannot bind = no recruitment of transcription factors
Interpret data as it relates to the lac operon and other similar methods of regulation
see clicker questions: I = gene for repressor O = operator; regulatory sequence Z = B-gal + = functioning normally; wild type - = nonfunctional protein made (loss of function mutation) c = constitutive; mutation; constantly produced (only affects O)
Predict how DNA methylation patterns and histone acetylation will affect gene expression
see clicker questions: - methylation shuts off gene expression - acetylation increases gene expression
Explain how restriction enzymes recognize and cut DNA
techniques for cutting DNA depend on specific DNA sequences because the enzymes only recognize, attach, and cleave at certain sites. cutting DNA allows whole genomes to be broken up into smaller pieces for further analysis like DNA sequencing. restriction sites are typically four or six base pairs long. Whenever a restriction enzyme finds a restriction site, it cleaves the nucleotides at the same spot on the antiparallel top and bottom strands. This symmetry is palindromic (it reads the same in both directions). the site of cleavage is not in the center of the recognition sequence. The cleaved double-stranded molecules therefore each terminate in a short single-stranded overhang.
Explain how DNA damage and/or mismatches are detected and repaired during base-excision repair, nucleotide-excision repair, mismatch repair, and double-strand DNA break repair (non-homologous end joining and homologous recombination)
Mismatch repair: -Fixes mispairing of bases during DNA replication, due to error of DNA polymerase -Post-replication repair: enzyme recognizes error, recruits protein that cleaves DNA backbone, exonuclease removes nucleotide and DNA poly fills it in. Ligase joins backbones. Base Excision: -Corrects abnormal bases, due to carinogens or hydrogen peroxide, whose addition of bulky side groups prevent proper base bonding Steps: -Abnormal base is cleaved from sugar in DNA backbone -Baseless sugar is removed from backbone -Repair poly inserts correct nucleotide into the gap Nucleotide Excision Repair: -Repairs short stretches of DNA containing mismatched pairs or damaged bases due to UV light, which causes cross links between pyrimidine bases -Steps: -Instead of degrading a DNA strand nucleotide by nucleotide until mismatch is removed, it removes an entire damaged section of strand at once -Gap is filled by new DNA synthesis, using ungapped strand as template Double Stranded DNA break repair: -Repairs DNA when two strands are broken, due to gamma rays and x/rays -Homologous Recombination Uses an undamaged section of similar DNA as template Enzymes interlace undamaged strands and damaged strands and get them to exchange sequences -Nonhomologous End Joining Series of proteins trims off few nucleotides and fuses broken ends together Not accurate but useful when sister DNA is not available
Determine the appropriate DNA repair pathway that a cell would use in response to a particular type of DNA mutation or damage
Mismatch repair: A mispairing of one base Caused by DNA polymerase Base Excision repair: When a base is damaged (just one) carcinogens or hydrogen peroxide Nucleotide Excision Repair: When short stretches of DNA are damaged UV light Homologous Recombination: 2 broken strands and sister DNA is available Nonhomologous End Joining: 2 broken strands and no sister DNA available (not as accurate)
Identify different sources of DNA mutation and damage
Mistakes in DNA replication unrepaired damage to DNA, caused by: reactive molecules produced in normal course of metabolism Chemicals in environmental (carcinogens) Radiation (X-rays/UV light) Infectious agents (HPV, heliobacter pylori) Incorrectly repaired chromosome breaks Transposable elements - which move from one location to another in a DNA molecule and have the capacity to disrupt the normal function of a gene
Describe how DNA methylation affects gene expression in eukaryotes
More methylation = less access for promoter to bind to DNA sequence It decreases gene expression because it represses transcription by condensing chromatin.
Predict how changes in telomerase activity affect cell division
More telomerases = more telomeres = more cell division Less telomeres = less cell division
Describe how mutations arise and how environmental factors can increase mutation rate
Mutations are random; they are not directed by the environment. This does not mean that the environment cannot affect the rate of mutation. Mutagens increase the rate of the mutation, but they cannot induce specific mutations that would be beneficial to the organism in response to the environment.
Design an appropriate primer pair to amplify a given region of DNA using PCR
Needs to match beginning of sequence that you want to amplify moving in 5' to 3' direction Ex: 5' TCAAACTTGC.....GTGGCACAAA 3' 3' AGTTTGAACG....CACCGTGTTT 5' 5'-ACTTGC-3' and 5'-TGCCAC-3'
Define the terms oncogene, proto-oncogene, and tumor suppressor
Oncogene - a cancer causing gene Proto-oncogene - the normal cellular gene counterpart to an oncogene which is similar to a viral oncogene but can only cause cancer when mutated Tumor suppressor - a family of genes that encode proteins whose normal activities inhibit cell division P35
Define and relate the terms operon, polycistronic mRNA, and structural gene
Operon - region of DNA containing promoter, operator, and coding sequence for structural genes Polycistronic mRNA - when coding sequences of structural genes are transcribed together into a single molecule of mRNA Structural gene - genes that code for the sequence of amino acids making up the primary structure of each protein, code for the primary structure of proteins
Identify the differences between positive and negative forms of regulation
Positive = induces transcription to occur through binding of an activator to its operator; usually this occurs for proteins needed only when small molecule is in the cell (e.g. need to break it down) Negative = represses transcription from occurring through binding of a repressor; usually occurs in genes that are needed for the synthesis of a molecule (to prevent too much product from being made, product is its allosteric repressor)
Describe the role of posttranslational protein modifications in the control of gene expression
Post translational protein modifications are processes that modify proteins in multiple ways that regulate their structure and their function: For example, adding a sugar molecule to the side chains of some amino acids can alter a protein's folding or target the molecule to certain cellular components In addition, adding phosphate groups to amino acids introduces a negative charge and dramatically alters the conformation of the protein posttranslational modification - mod. after translation of proteins in ways that regulate their structure/function ex: phosphorylation, methylation, acetylation
