Exam 1
Explain how certain micro satellite sequences can be used to obtain an individual's genetic fingerprint (paternity testing, forensic analysis)
4e. Micro satellite sequence can be used to obtain an individual's genetic fingerprint for forensic analysis. For example, three different micro satellite regions may be amplified by PCR and visualized through agarose gel electrophoresis to place a suspect at a crime scene. The number of repeats is proportional to the size of the PCR product (since the primers flank the repeat sequence). To determine the number of repeats, subtract the size of the primer from the size of the PCR product and divide that by the size of the repeat.
Sulfur amino acids
Cysteine, methionine
Indicate how domains are covalently linked in a multi-domain protein
Domains are covalently linked in a multi-domain protein through loops and turns in the amino acid sequence
Define a domain
Domains are self-folding structures found in many proteins; they are constituents of multi-domain proteins and can serve a specific function such as calcium binding
Describe how peptide bond resonance hybridization can account for important properties of the peptide bond such as planarity, polarity, resistance to rotation, and tendency to form hydrogen bonds
Electrons migrate from the C-O double bond to give the C-N single bond double bond character, causing the oxygen to have a partial negative charge. This partial negative charge on the oxygen atom increases its tendency to hydrogen bond. The partial negative charge on the oxygen atom also creates a partial positive charge on the nitrogen atom, increasing the bond polarity. Furthermore, the bond is planar and the double bond character prevents free rotation. This bond is typically found in the trans orientation in which the alpha carbon is up on once side of the bond and down on the other side of the bond
Explain positive cooperativity in oxygen binding to hemoglobin
Positive cooperativity occurs when the binding of oxygen to one subunit increases adjacent subunits' affinity for oxygen. Similarly, the unloading of oxygen from one subunit decreases adjacent subunits' affinity for oxygen.
Understand the relationship between somatic and germline mutations
a. Somatic mutations are passed on to only the daughter cells of the mutated somatic cell; this may result in cancer. Germline mutations will be present in all the cells that arise from the mutated gamete.
Describe the functions of the F8 proximal histidine and the E7 distal histidine
The F8 proximal histidine forms a covalent coordination with iron, while the E7 distal histidine hydrogen bonds with oxygen once it has been bound.
DNA cloning
The ability to select a specific nucleic acid sequence from all the sequences in a cell and produce it in a purified or enriched form
What does the complexity of eukaryotic genomes result from?
The abundance of different types of coding and non coding DNA sequences
Define catalysis
The act of speeding up a reaction
Describe how the initial rate of an enzyme-catalyze reaction changes with increasing substrate concentration
The initial rate of an enzyme-catalyze reaction increases with increasing substrate concentration; the curve is logarithmic
Explain the role of the water molecule that is bound to the active site zinc ion of CPA
The negatively charged oxygen on Glu270 attacks a hydrogen atom in the water molecule that is bound to the active site zinc ion of CPA, which results in the oxygen atom in the water molecule to attack the carbonyl carbon involved in a peptide bond. Several other exchanges of electrons occur, eventually resulting in the cleavage of the peptide bond.
Tertiary protein structure
Includes the total 3-dimensional structure of a protein including side chain bond angles (this is the highest structure for a protein composed of only subunit
Basic amino acids
Lysine, arginine, histidine
Eukaryotic chromosomal DNA replication differs from prokaryotic DNA replication in what three ways?
1. It is compartmentalized within the nucleus, partitioning it from the site of synthesis of replication proteins and precursors and from extra cellular stimuli that may trigger initiation of synthesis 2. It begins at multiple origins, activated throughout S phase in a precise and temporally regulated manner 3. The nucleosomal proteins must be duplicated along with the DNA to maintain proper chromosomal organization
DNA excision repair mechanisms have what four steps in common?
1. Recognition of damage 2. Cleavage by endonuclease 3. Removal by nuclease or helicase activity 4. Replacement by polymerase and ligase activity
Describe mutations in the beta globin gene and indicate how they can give rise to beta thalassemias
4h. One splicing mutation that causes beta thalassemia changes the G in GU to an A, destroying the 5' splice site and activating one of three cryptic 5' splice sites that result in either the removal of part of exon 1 or the inclusion of part of intron 1. These mutations cause a frame shift mutation that is likely to result in an in frame stop codon and thus a short protein. Another splicing mutation (C—>G) in intron 2 creates a new 5' splice site and activates an adjacent cryptic 3' splice site. This results in the incorporation of part of intron 2 in the final protein product.
Predict the molecular consequence of mutations in NER factors on the NER pathway
7d. Mutations of the XPA-XPG; XPV (at least eight) genes involved in NER results in xeroderma pigmentosum (XP); this disease presents with sun sensitivity, cancer, and neurological defects. Mutations of the CSA; CSB genes involved in TCR results in Cockayne syndrome (CS); this disease presents with sun sensitivity, post-natal developmental defects, neurological defects, and premature aging.
Explain what a Hill coefficient indicates about cooperativity
A Hill coefficient of 1 indicates that there is no cooperativity. A Hill coefficient more than 1 indicates that there is positive cooperativity. A Hill coefficient less than 1 indicates that there is negative cooperativity (for example, need greater than a 81-fold change in oxygen concentration to go from 10% saturation to 90% saturation).
Describe the basic effect of a competitive inhibitor on enzyme activity
A competitive inhibitor is a molecule that closely resembles the substrate and binds to the active site of the enzyme; as the name implies, it forces the substrate to compete for its place in the active site
Describe the type of reaction that is catalyze by carboxypeptidase A (CPA) and indicate whether or not this reaction could occur without an enzyme
Carboxypeptidase A (CPA) is a hydrolase, so it uses water to make two products from a singular substrate ("break with water"). Specifically, it removes amino acids from the C-terminus of peptides. This reaction could NOT occur without an enzyme - it would take over 100 years to complete!
Understand that DNA polymerase has editing (3' to 5' exonuclease activity) as well as polymeric in function, why this is important for high fidelity replication, and potential impact of mutations in the proofreading domain of DNA polymerase
DNA polymerase has editing (3' to 5pm exonuclease activity) in addition to its polymeric in function. This contributes to the overall high fidelity of replication, which is about one error in one billion nucleotides. Germaine mutations affecting the DNA polymerase proofreading domains predispose individuals to colorectal adenomas and carcinomas (for example, Leu424Val in POLE and Ser478Asn in POLD).
Assess the unique properties of DNA polymerases
DNA polymerase requires a 3' hydroxyl group to synthesize DNA (hence, why synthesis only occurs in the 5' to 3' direction). DNA polymerase requires an RNA primer to begin synthesis. DNA polymerase is only loosely bound to the template strand while it is synthesizing. DNA polymerase binds to a regulated sliding clamp (PCNA), which aids in its recruitment and movement along the template strand. DNA polymerase had two active sites, one that polymerizes and one that edits.
What are genes?
DNA sequences that produce a functional RNA molecule (encodes a protein or forms a structural or regulatory RNA)
What structural features do vassopressin have?
Disulfide bond and a C-terminal, uncharged amide group
Lysine methylation
Does not affect the change of the residue, it results in the recruitment of different protein complexes.
Explain mechanisms for dynamic changes in chromatin structure
Dynamic changes in chromatin structure are achieved via ATP-dependent chromatin remodeling complexes. These complexes pull on the DNA, allowing the histone to slide along the DNA. These complexes (with the help of histone chaperones) can also exchange H2A-H2B dimers within a histone as well as an entire nucleosome core (histone octamer); the modified dimer or histone variant may make the DNA more or less accessible by proteins.
Understand the roles of histone H1 and inter-nucleosome tail interactions in chromatin compaction
Histone H1 and inter-nucleosome tail interactions play a role in the compaction of nucleosomes ("beads on a string") into the 30 nm fiber (zig zag model of chromatin organization). Histone H1 attaches to the DNA where it enters or exits the histone, and the inter-nucleosome tail interactions occur between histone tails and adjacent histone cores.
Role of histone variants
Histone variants are histone proteins that vary slightly from the parent histone protein (usually at the C-terminal). These variants are typically associated with a specialized function. For example, CENP-A is an H3 variant resulting from an insertion that plays a role in centromere function and kinetochore assembly. All histone proteins except H4 have variants.
Describe homology modeling as a technique for predicting the tertiary structure of a protein from the amino acids sequence
Homology modeling is a technique used to predict the tertiary structure of a protein from the amino acid sequence. You search a structure database for a protein that has a similar amino acid sequence, do a sequence alignment, and use computer programs to "thread" the protein sequence through the X ray structure. In simpler terms, you use a known structure to approximate what the novel structure looks like.
Indicate some situations where BPG in red blood cells is increased and how this increase is beneficial
BPG in red blood cells is increased during times of hypoxia; this can occur in an individual with anemia or lung diseases such as COPD or at high elevations. BPG binds to the beta-beta interface of deoxygenated (T) hemoglobin, which stabilizes the conformation and forces equilibrium to the left in the above reaction once again. Delivering oxygen to the tissues is more easily carried out with hemoglobin in the T conformation.
Indicate which optimal isomer is found in proteins
L amino acids are found in proteins
Explain why lactic acid and carbonic acid drive hemoglobin to the T conformation and enhance oxygen delivery to tissues
Lactic acid and carbonic acid release protons, which drive the above reaction to the left in order to achieve equilibrium. This produces more hemoglobin in the deoxygenated (T) conformation, which promotes the release of oxygen at the tissues.
Describe large conformational changes a using hexokinase as an example
Large conformational changes can occur when a substrate enters an active site; maybe one or more domains will fold over another portion of the protein.
Describe what processes cause most of the alpha thalassemias and beta thalassemias
Most alpha thalassemias are caused by alpha globin gene deletion; in this case, hemoglobin is made up of four beta subunits, which cannot release oxygen. Most beta thalassemias are caused single base pair mutations in the beta chain, leading to decreased or defective beta chain mRNA
Oxidoreductase
Moves electrons, hydrogen atoms, or oxygen atoms from one substrate to another
Predict the consequence of mutating a glycine to another amino acid in collagen
Mutating a glycine to another amino acid in collagen would greatly impact the protein's structure because there is simply not room for any other side chain in the interior of the triple helix. Since structure determines function, the function of collagen would be compromised as well
Indicate whether or not mutations in a protein automatically lead to loss of protein function
Mutations in a protein do not automatically lead to loss of protein function; in some cases, they can lead to gain of protein function, whether it is simply more active than it usually is or it acquires a new function altogether
Compare myoglobin and hemoglobin with respect to the fold change in pO2 necessary to go from 10% saturation to 90% saturation
Myoglobin requires an 81-fold change in the concentration of oxygen to go from 10% saturation 90% saturation. Hemoglobin requires just a 4.8-fold change in the concentration of oxygen to go from 10% saturation to 90% saturation (this is because of positive cooperativity).
Holliday junction
What forms when the second strand is captured following an invasion event; resolution of this structure may or may not result in a crossover event
Describe the consequences of the beta chain Phe42Ser mutation in Hb Hammersmith
Phenylalanine 42 locks the heme in place once it enters the heme binding pocket. When phenylalanine 42 is changed to serine, the heme is no longer locked into place by the serine and slips out of the pocket. As a result, hemoglobin is unstable and precipitates. This condition is known as hemoglobin Hammersmith.
Detection of an RNA target by PCR
Prepare a cDNA copy of the RNA and use the cDNA as a template for PCR
RNA pol II
RNA polymerase II: transcribes mRNA, snoRNA, miRNA, siRNA, most snRNA RNA polymerase II: promoters include TATA, CAAT, BRE, DPE, and others
Describe how NGS is performed
a. NGS is performed by attaching a Fluor to a nucleotide (chain terminator with 3' hydroxyl block), which is incorporated into the growing chain by DNA polymerase. A photo is taken, which is followed by the removal of the Fluor and the block. This repeats with the addition of the next nucleotide until all of the DNA fragments are sequenced. Essentially, it sequences one nucleotide at a time with each photo taken.
Compare the mechanisms for repair of DNA double strand breaks
a. NHEJ: double strand break is recognized by Ku heterodimers, which recruit additional proteins; these proteins chew back the sticky ends to create blunt ends that are processed, repaired, and ligated. The repaired DNA usually suffers a deletion of nucleotides; this is typically tolerated because most of the genome is non-coding. HEJ: nucleases digest the 5' ends of the broken strands; the 3' end of one of the broken strands invades the sister chromatid by complementary base pairing (this reaction is catalyze by RecA/Rad51 proteins); repair polymerase synthesizes DNA using unmanaged DNA as a template; the invading strand is released (with the help of RubA/RubB proteins), and DNA synthesis continues using strands from damaged DNA as a template; nicks are ligated
Describe the primary advantage of a microarray experiment compared to other hybridization techniques such as Northern blotting or RT-PCR
a. Northern blotting and RT-PCR are time-consuming, expensive, and require lots of a sample (this is okay in the case of urine and blood but no so much for a small biopsy). Microarrays are advantageous because they can measure the expression of 20-250,000 genes simultaneously.
RNA pol I
a. RNA polymerase I: transcribes rRNA (5.8S, 18S, 28S rRNA genes) c. RNA polymerase I: promoters include ribosomal initiator element, upstream promoter element (UPE)
Describe how PCR exponentially amplifies a PCR product
a. The PCR components include the DNA sample, primers that are complementary to the region flanking the DNA that you want to amplify, nucleotides, DNA polymerase, and buffer. These components are put into a PCR tube, which is placed in a thermal cycler. The thermal cycler rotates through three temperatures — 95 degrees Celsius for denaturation, 55 degrees Celsius for annealing (primers bind to DNA), and 72 degrees Celsius for extension (DNA polymerase synthesizes the new strand) — 20 to 40 times to yield millions of copies of the DNA sequence of interest.
Explain the "end replication" problem
a. The end-replication problem is that the 3' end of the template DNA has no room for a primase to synthesize a RNA primer, leaving a gap at the end of the lagging strand.
Identify key features of tRNA genes that direct their transcription
a. The pre-tRNA gene uses two promoters: A-box and B-box. TFIIIC binds the B-box with high affinity and the A-box with low affinity. Then, TFIIIB (composed of TAFs and TBP) binds, and RNA polymerase III is recruited. The pre-5S-rRNA gene uses two promoters: A-box and C-box. TFIIIA binds the C-box first, followed by the steps outlined for the pre-tRNA gene.
Transcription
a. Transcription: the process that converts DNA into RNA; occurs in the nucleus
Describe the types of diseases that might be amenable to gene therapy
a. Types are diseases that might be amenable to gene therapy include genetic disorders (replacing a mutated gene with a normal copy, inactivating a mutated gene that functions improperly) and other diseases (introducing a new gene to help fight the disease).
Relate various DNA lesions with the type of repair mechanisms
a. X-rays, oxygen radicals, alkylating agents, spontaneous reactions —> uracil, abasic site, 8-oxoguanine, single-strand break —> base excision repair (BER) UV light, polycyclic aromatic hydrocarbons —> pyrimidine dimers, bulky adduct —> nucleotide excision repair (NER) X-rays, anti-tumor agents —> inter strand cross link, double-strand break —> recombinational repair Replication errors —> A-G mismatch, T-C mismatch, insertion, deletion —> mismatch repair
Describe the primary differences between DNA and cDNA
a. cDNA is complementary to mRNA, which makes it complementary to the DNA that made the mRNA product.
Nucleic acid synthesis is governed by what four important characteristics?
1. A pre-existing nucleic acid strand is copied of Watson-Crick pairing 2. Nucleic acid strands grow in only one direction (5' to 3') 3. Polymerases synthesize nucleic acids 4. Duplex DNA synthesis requires a special growing fork because the strands are anti parallel
Explain the main differences between global NER and TCR
7e. The difference between global NER and TCR is in the recognition step. In TCR, the damage must be recognized by RNA polymerase during active transcription. When damage is recognized, RNA polymerase stalls and backs up at the damage site (prolonged arrest can lead to apoptosis). In global NER, XP proteins recognize non-specific bulky lesions at any point in the DNA and at any point in the cell cycle. Another difference is that TCR involves CSA and CSB proteins, which are associated with RNA polymerase during recognition. The pathways converge at the cleavage and removal steps, which involve TFIIH, DNA polymerase, and DNA ligase.
Define micro satellite sequence
4d. A micro satellite sequence is a series of short tandem repeats of DNA; they are typically 2-6 base pairs in length, occur in non-coding regions, and vary by individual.
Important signals in mRNA that direct splicing
5' splice site: GU branch point: A 3' splice site: AG
Explain how an enzyme greatly increases the rate of a chemical reaction
By stabilizing the transition state
Intron
Found in genes (coding regions of DNA), transcribed, and excised from the mRNA
What double strand break repair mechanism is preferred in S/G2?
HEJ
Describe the two components of heme and the basic shape of heme
Heme is made up of a ferrous iron bound to four nitrogens in carbon rings. One side of the heme is non-polar, and the other side of the heme is polar (it contains two carboxylic groups).
Heterochromatin
Highly folded DNA; contains no genes (centromeres and telomeres) or is resistant to gene expression; dynamic and self-sustaining once formed
List the forces that stabilize a folded protein and describe how they work
Hydrogen bonding occurs between a lone pair of electrons on N, O, or F and a hydrogen attached to N, O, or F. Ionic bonds are attractive and repulsive electrostatic interactions that occur between charged atoms. Van der Waals interactions occur when a spontaneous dipole, or separation of charge on one atom induces a dipole, or separation of charge on another atom; the partial charges are then attracted to each other. Hydrophobic interactions occur between hydrophobic side groups, and they serve to stabilize the interior of a protein by minimizing interaction with water (this interaction is favorable and increases the entropy of a system).
Describe the positioning of H-bonds that stabilize the alpha helix
Hydrogen bonding occurs in the direction of the helix between carbonyl oxygens and amino nitrogens
Define hydrophobic and hydrophilic as it pertains to amino acid side chains
Hydrophobic is synonymous with non-polar; hydrophobic side chains do not interact with water and are most commonly found on the interior of a folded protein, interacting with other non-polar side chains. Hydrophilic means water-loving; these side chains are polar and sometimes charged, and they will interact with water forming hydrogen bonds; hydrophilic side chains are usually found on the surface of proteins
DNA sequencing
Identifies the bases of a DNA fragment; essentially a modified PCR reaction with a single primer where labeled nucleotides are inserted into the DNA fragment as it is replicated
Define identity and similarity as it applies to sequence alignment of two proteins
Identity means that both amino acid sequences have the same amino acid in a given position. Similarity means that the amino acid sequences have amino acids with similar physical and chemical properties in a given position (for example, a glutamate and an aspartate or a lysine and an arginine).
Predict the relative pI values for two proteins based on their amino acid compositions
If a protein contains more acidic amino acids than basic amino acids, its isoelectric point will be less than pH 7. If a protein contains more basic amino acids than acidic amino acids, its isoelectric point will be treated than pH 7.
Explain how an imbalance in alpha chains and beta chains can lead to anemia
If there is an imbalance of alpha chains and beta chains, the excess subunit precipitates and destroys the red blood cells (anemia).
Describe the method of agarose gel electrophoresis
In agarose gel electrophoresis, plasma proteins are separated by charge. Most proteins are negatively charged at pH 8.6 and will thus migrate towards the anode
Explain the relationship between cysteines and cystine in protein structures
A cystine disulfide bond is formed from two cysteines within the primary structure of a protein (this is an oxidation reaction in which two hydrogens are lost). Cystine bonds to act to stabilize the native structure of a folded protein
Define derived amino acid
A derived amino acid refers to an amino acid that has been altered after translation
Describe how a non competitive inhibitor affects enzyme activity
A non-competitive inhibitor affects enzyme activity by binding to a portion of the enzyme that is NOT the active site in a way that disrupts the correct positioning of amino acids in the active site; the substrate can still bind to the active site, but the enzyme-substrate-inhibitor complex is nonproductive
Lyase
Cleave C-C, C-N, or C-O bonds
Restriction enzyme
Cleave double-bonded DNA at specific sequences
Describe the structure of collagen
Collagen is a three-chain super helix (a helix of three helixes). There are three amino acids per, and it is two times more extended than an alpha helix.
Provide an approximate definition of peptide and protein
A peptide is fewer than 50 amino acids; a protein is greater than 50 amino acids
Heteroduplex joint
A segment of DNA where strands from two different DNA helixes have base paired
PCR
A sensitive molecular technique that amplifies a specific nucleic acid sequence within a complex nucleic acid mixture; exponentially amplifies the sequence to generate millions of copies that are readily detectable by gel electrophoresis
Define a fold
A specific arrangement of secondary structure elements; examples include all alpha helix globin fold, central beta barrel, twisted best sheet, and beta barrel jelly roll
Lysine acetylation
Decreases positive charge (which is how the protein interacts with the DNA), resulting in a less tight interaction with DNA.
Indicate whether or not deltaG predicts the rate of the reaction
DeltaG does NOT predict the rate of a chemical reaction
Define deltaG and indicate when this term predicts a spontaneous chemical reaction with the release of energy
DeltaG is the change in Gibbs free energy for a chemical reaction. A negative deltaG predicts a spontaneous chemical reaction with the release of energy.
Define denaturation
Denaturation is when a protein loses its secondary, tertiary, and/or quaternary structure through the weakening of hydrogen bonds, ionic bonds, van der Waals interactions, and hydrophobic interactions
Distinguish different types of DNA sequences
Different types of DNA sequences include genes (protein-coding regions and introns), other unique sequences (non-repetitive DNA that is neither in introns nor codons - may serve a regulatory purpose), repeated sequences (simple sequence repeats and segmental duplications), and transposons (LINEs, SINEs, retroviral-like elements, and DNA-only transposon fossils).
Southern blotting
A technique for detection and quantifying a specific DNA within a complex DNA mixture
Northern blotting
A technique for detection and quantifying a specific RNA within a complex RNA mixture
Western blotting
A technique for detection and quantifying a specific protein within a complex protein mixture
Next generation sequencing (NGS)
Allows millions of small DNA fragments to be sequenced at the same time (termed high throughput) compared to a single DNA sequence in traditional sequencing
RT-PCR
Allows us to determine which genes are being transcribed in a cell; also allows for the detection of RNA viruses such as HIV and influenza A
Define kcat
Also known as the turnover number; the number of substrate molecules converted to product per enzyme per second; measured in units of sec-1
How are amino acids joined?
Amino acids are joined by a peptide bond that connects the carboxylic acid group of one amino acid with the amino group of a second amino acid.
Indicate whether an X-ray structure can always reveal the structure of the active protein
An X-ray structure cannot always reveal the structure of an active protein, especially if it has unstructured portions (those that are loose and just fly in the breeze).
Indicate whether or not enzyme amino acids located at a distance from the active site are important for substrate binding and catalysis
Enzyme amino acids located at a distance from the active site are important for substrate binding and catalysis because they may be involved in protein folding that brings the active site amino acids together and/or allows for the correct orientation of the substrate within the active site.
Acid amino acids and their derivatives
Aspartate, asparagine, glutamate, glutamine
Indicate whether or not loops and turns can be categorized as regular secondary structure
Loops and turns cannot be categorized as regular secondary structures because the phi and psi bonds are not necessarily equal throughout the entire loop or turn
Cofactor
Metal ion that does not bind the active site (for example, zinc ion)
Cosubstrate
Modified during a chemical reaction and leave the active site (for example, ATP and NAD)
Indicate whether or not most chemical reactions in the body can take place at physiological conditions without a catalyst
Most CANNOT take place without a catalyst
Isomerase
Move a group or double bond within a molecule to create an isomer
Describe the overall secondary structure of myoglobin and hemoglobin chains
Myoglobin is a monomer consisting of 153 amino acids organized into several alpha helixes connected through corners, loops, and bends. Hemoglobin is a tetramer consisting of two identical alpha chains and two identical beta chains; each subunit is organized into alpha helixes connected through corners, loops, and bends. Myoglobin and the beta chain of hemoglobin look very similar. Myoglobin and each subunit of hemoglobin contain a heme prosthetic group (non-polypeptide moiety) that binds oxygen
Where does the biogenesis of ribosomes take place?
Nucleosome
Describe the components and the structural organization of the nucleosome
Nucleosomes (comprised of DNA and histone proteins) are the basic subunit of DNA organization. Each nucleosome is composed of 147 base pairs wrapped tightly around a core histone. Adjacent nucleosomes are connected through linker DNA. Each nucleosome has a diameter of about 11 nm. The octameric histone core is made up of two of each of the following: H2A, H2B, H3, and H4. H3 and H4 form dimers, which form a tetramer (core); H2A and H2B form dimers (earmuffs). There are 142 hydrogen bonds between DNA and the histone core. Portions of histones, known as histone tails, protrude through the nucleosome. DNA is wrapped 1.7 turns around each nucleosome. Nucleosomes often resemble "beads on a string."
Crossover
Occurs when homologous chromosomes exchange information
Describe Okazaki fragments and their function
Okazaki fragments are about 100-200 nucleotides long; they are synthesized along the lagging strand, since DNA polymerase can only synthesize in the 5' to 3' direction. Each Okazaki fragment is begun with an RNA primer.
Define an amphipathic alpha helix
One in which charged/polar side groups are found on one side of the helix and non-polar side groups are found on the other side of the helix
Coenzyme
Organic molecule that binds loosely to the active site and aids in substrate recruitment
What three types of DNA sequences that are required to produce a eukaryotic chromosome that can be replicated and then segregated at mitosis?
Origin of replication, centromere, and telomeres
Indicate the trigger for the conformational change that occurs upon oxygen binding
Oxygen binding pulls the iron down to the plane of the heme molecules, which sets off a cascade of movements throughout secondary structures (alpha helixes).
Describe the unique structural feature of proline
Proline is a cyclic amino acid. The amino terminus nitrogen forms a covalent bond with the end side chain carbon, forming a 5-membered ring
Promoter
Promoter: a regulatory region in a gene that recruits basal transcription factors to initiate transcription; multiple promoters (2-3) work together in eukaryotic cells (this means a defect in one promoter is NOT catastrophic)
Indicate which conformation has the highest affinity for oxygen
R conformation
Describe termination of mRNA transcription in eukaryotic cells
RNA polymerase II: can terminate at multiple sites located over a distance of 0.5-2kb beyond the polyA addition site; there is no clear termination sequence or structure
RNA pol III
RNA polymerase III: transcribes tRNA, rRNA (5S), some snRNA, other small RNA RNA polymerase III: promoters include A-box/C-box (5S rRNA) and A-box/B-box (tRNA)
Gene therapy
Refers to the experimental manipulation of genes for the treatment or prevention of disease
Strand invasion
Refers to the invasion of a sister chromatid following a double stranded break
Primary protein structure
Refers to the sequence of amino acids and the location of disulfide bonds
Secondary protein structure
Refers to the shape of the polypeptide backbone in a folded protein (includes alpha helixes and beta sheets); does not include side chains
Exon
Regions of a gene that are transcribed and determine the amino acid sequence of the mature protein product
Define regular secondary structure
Regular secondary structure means that all of the phi and psi angles are equal (they repeat over and over again)
Indicate what is most commonly affected by disease-associated mutations
Regular secondary structure, tight turns, and bends
What is the level of chromatin organization in interphase?
Relatively extended form (30nm fiber)
Direct repair
Reverse base damage; energetically costly to cells and used to quickly repair highly mutagenic lesions
Explain how the rotation of covalent single bonds in a polypeptide chain backbone can generate secondary structure
Rotation of covalent single bonds in a polypeptide chain backbone can generate secondary structure by causing regularity in the phi and psi bonds. This results in alpha helixes and beta-pleated sheets
Hydroxyl amino acids
Serine, threonine
Describe the geographical distribution of the sickle cell trait and the reason for this distribution
Sickle cell trait is prevalent in regions in the Middle East, Africa, and Asia because it provides a protective factor against malaria. The malaria parasite cannot complete its life cycle in the red blood cells of individuals with sickle cell trait.
Describe the difference between sickle cell trait and sickle cell disorder
Sickle cell trait is the term applied to an individual who is heterozygous (1 normal beta chain, 1 mutated beta chain). Those with sickle cell trait do not experience symptoms. Sickle cell disease is the term applied to an individual who is homozygous for the disease (2 mutated beta chains). Those with sickle cell disease do experience symptoms.
Indicate how amino acid side chains are oriented in a beta sheet
Side chains will be orientated above and below the beta sheet
What is a likely mechanism for triplet repeat expansion?
Slipped mispairing
Splicing
Splicing: the process that results in the removal of introns from an mRNA
Indicate the geographical distribution of thalassemia and why heterozygotes have a survival advantage
Thalessemia traits are common in the Middle East, Africa, and Asia because heterozygotes are resistant to malaria.
Explain the Central Dogma of Molecular Biology
The Central Dogma of Molecular Biology states that DNA is transcribed into mRNA in the nucleus; the mRNA is then transported to the cytosol, where it is translated into a protein
Indicate which chains are most highly expressed in the fetus
The alpha and gamma chains are the most highly expressed in the fetus.
Describe what happens when an alpha helix is followed by a Pro-Gly sequence in a polypeptide chain
The alpha helix will be terminated and a beta sheet will be started in the direction about perpendicular to the alpha helix
Describe the physiological properties of the heme binding pocket
The amino acid side chains of hemoglobin that make up the heme binding pocket are hydrophobic. The pocket is between the E and F helixes.
Quaternary protein structure
The arrangement of sub units through non-covalent interactions in a multi-subunit protein
Define initial rate
The change in product over time at the start of a chemical reaction when there is excess substrate and little to no product
Understand the concept of histone writers and readers
The combination of histone tail modifications attracts specific reader and writer proteins, which execute appropriate functions. Writer proteins are the proteins that make the histone tail modifications, which attract reader proteins. Reader proteins bind to the modified histone tail, which attracts a protein complex with catalytic activities (another writer protein) to modify an adjacent histone tail. This series of events can be repeated over and over again, resulting in either chromatin condensation (to silence a gene) or de-condensation (to activate a gene). I
pO2
The concentration of oxygen given as "partial pressure" in units of torr
P50
The concentration of oxygen required for 50% of myoglobin or hemoglobin to have oxygen bound
Describe the concentrations of substrate and product change over time
The concentration of substrate is going to decrease over time, while the concentration of product will increase over time
Describe how proline affects protein structure
The double bond character in a proline-containing peptide bond is reduced, allowing slight rotation about the peptide bond. However, there is no rotation about the phi bond. Proline will decrease secondary structure by causing a kink in an alpha helix or beta-pleated sheet.
Ki
The enzyme-inhibitor dissociation constant; it is a measure of the affinity of an inhibitor to its enzyme
Define transition state
The highest energy intermediate
Histone code
The histone code refers to the idea that certain modifications or combinations of modifications have different meanings. For example, methylation at K9 results in gene silencing, whereas acetylation at K9 in conjunction with methylation at K4 results in gene expressions
Describe how hydrogen bonds are orientated in a beta sheet
The hydrogen bonds are perpendicular to the direction of the amino acid strands
Sister chromatid
The identical chromosomes joined at the centromere
Identify key features of rRNA genes that direct their transcription
b. RNA polymerase I uses two promoters: upstream control element (UCE) and core element (core). These promoters must work because there are no back-ups like there are for mRNA transcription. Upstream binding factor (UBF) binds and dimerizes on the DNA, which forms a loop in the DNA that recruits SL-1 (composed of TAFs and TBP) and RNA polymerase I.
Explain how recombinant DNA can be used in clinical medicine
b. Recombinant DNA can be used to create protein products for use in clinical medicine. For example, recombinant DNA encoding insulin can be inserted into a plasmid. The recombinant plasmid is then introduced into E. coli in a way that forces the bacteria to produce insulin (contains promoter sequence). The insulin is then harvested from E. coli and uses in the treatment of diabetes.
Identify the types of DNA ends generated by different restriction enzymes
b. Restriction enzymes can either produce blunt ends (no overhangs) or sticker ends (overhangs of various length).
Describe the mechanism for the addition of telomere repeat sequences
b. Telomere repeat sequences are added to the 3' end of the lagging strand by telomerase. Telomerase uses an RNA template rich in guanine to elongate the 3' end (this is known as RNA-templates DNA synthesis). The elongated 3' end allows the addition of an RNA primer and the attachment of DNA polymerase to complete synthesis.
Describe the major differences between NGS and traditional sequencing
b. The major difference between NGS and traditional sequencing is that NGS is much more powerful — you get >10^6 reads per array, whereas, with traditional sequencing, you get only one read per capillary.
Describe and assess cell controls that assure that each region of eukaryotic DNA is replicated only once per cell cycle and that all DNA is replicated before onset of mitosis
b. The phosphorylation of origin recognition complex (ORC) prevents the re-loading of additional DNA helicases following the recruitment of DNA polymerase, which ensures that each region of eukaryotic DNA is only replicated once.
Discuss the relative abundance of each type of RNA
b. rRNA is the most abundant type of RNA in the steady state as well as during RNA synthesis. tRNA is more abundant than mRNA in the steady state, but mRNA is more abundant that tRNA during RNA synthesis.
Describe the basic features of tRNA maturation
b. tRNA maturation involves splicing using a cut and paste method as well as hydrogen bonding within itself to form a cloverleaf shape
List a few potential applications for use of CRISPR/Cas in human therapies
e. CRISPR/Cas9 could be used in the clinical setting to eradicate monogenic hereditary disease, manipulate viral cellular targets, and produce immune therapies.
Describe the mechanism of red blood cell sickling and symptoms that result
In the deoxygenated state, hemoglobin has a normal hydrophobic patch on the surface of the protein consisting of alanine, phenylalanine, and leucine. When glutamate is changed to valine, valine forms hydrophobic interactions with this normal hydrophobic patch in the deoxygenated state. This causes the hemoglobin to precipitate into long polymers, which makes the red blood cell rigid. The diseased red blood cells get stuck in capillaries easily and block the passage of other red blood cells, preventing the oxygenation of tissues downstream. These diseased red blood cells have weak membranes that rupture as well, which can be painful.
DNA replication utilizes what six specialized mechanisms?
Initiation, unwinding, priming, unidirectional fork movement, untangling, and termination
Describe the basic structure of insulin
Insulin is a peptide hormone composed of two polypeptide chains. A chain has 21 chains, and B chain has 30 amino acids, resulting in 51 total amino acids. The two polypeptides are held together via two cystine bones (4 cysteines total); there is a third cystine bond within A chain
What is the level fo chromatin organization in mitosis?
It is most highly condensed (chromosome)
Ligase
Join atoms together using energy, usually from ATP
Indicate the mutation in sickle cell disease
The mutation in sickle cell disease is a missense mutation in which a glutamate is changed to a valine at amino acid position 6 in the beta chain
Explain the consequence of the mutation beta chain Asp99Asn in Hb Kempsey
The mutation in the beta chain, aspartate to asparagine at amino acid position 99 (FG corner), in hemoglobin Kempsey locks hemoglobin into the R conformation (high oxygen affinity) and prevents oxygen from being released in the tissues. This causes a cascade of events that results in overproduction of red blood cells.
Homologous chromosome
The non-identical set of chromosomes (one from mom and one from dad)
Describe the consequence of the structural change in the FG corner when oxygen binds
The structural change in the FG corner when oxygen binds alters the C helix of an adjacent subunit, increasing its affinity for oxygen.
Describe the two main classes of heminoglobinapthies
The two main classes of hemoglobinopathies are structural variants and thalassemias. Structural variants result from an altered amino acid sequence (usually a missense mutation), whereas thalassemias result from decreased abundance of one or more of the globin chains
In very general terms, explain how the active site zinc site and certain amino acids in the active site can stabilize the transition state
The zinc ion can stabilize the transition state by forming ionic interactions with the carbonyl oxygen involved in a peptide bond. Certain amino acids, especially negatively charged (as in the case of CPA with Glu270) and positively charged amino acids, can stabilize the transition state by forming covalent bonds and hydrogen bonding.
Indicate the number of amino acids in each turn of an alpha helix
There are 3.6 amino acids in each turn of an alpha helix
Indicate the prevalence of heminoglobinopathies compared to other genetic diseases
These diseases are the most common single-gene diseases (they result from a change affecting only one gene
Transferase
Transfer chemical groups, such as phosphates, from one molecule to another
Translation
Translation: the process that converts RNA to protein; occurs in the cytoplasm
snRNPs involved in splicing
U1: binds the 5' splice site U2: binds the branch point and forms part of the catalytic center U5: binds the 5' splice site; then the 3' splice site U4: masks the catalytic activity of U6 U6: catalyzes splicing *U1 binds first, followed by U2, and finally U5/U4/U6 *snRNPs base pair with the mRNA transcript and with one another
Hydrolase
Use water to make two products from a singular substrate
Nucleic acid hybridization
Uses a labeled, single-stranded DNA probe to detect a complementary nucleic acid sequence within a complex mixture
Microarray analysis
Uses hybridization techniques to monitor expression of thousands of genes simultaneously; powerful tools for research and personalized medicine
Branched chain amino acids
Valine, leucine, isoleucine
Define the T conformation and R conformation with result to oxygen binding
When oxygen is bound to hemoglobin, it is in the R conformation. When oxygen is not bound to hemoglobin, it is in the T conformation.
Function of mRNA
b. mRNA: messenger RNA, codes for proteins
List four issues or challenges that are currently faced by scientists developing new gene therapies
c. Challenges in gene therapy include how to deliver the gene (viral or non-viral vectors) and ensure that it activates the right cells, how to avoid an immune response in the case of a viral vector, how to prevent the disruption of important genes such as tumor suppressor genes in target cells, and how to make the therapy accessible at an affordable cost.
Describe how DNA sequencing can be used in the diagnosis or treatment of disease
c. DNA sequencing can be used in the diagnosis of disease by allowing you to compare an individual's gene sequence to known mutations of the suspected disease. For example, cystic fibrosis can be diagnosed following discovery of the deleted phenylalanine (Phe508) in the CFTR gene sequence of an affected individual.
Explain how RT-PCR might be useful in the diagnosis of disease
c. RT-PCR may be useful in the diagnosis of disease caused by RNA, such as HIV.
Analyze the relationship of telomere length with cellular life span
c. Telomere length is inversely related to cellular life span (the longer the cell life/the more it undergoes replication, the shorter its telomeres).
Explain how the information generated from a microarray might be utilized to better understand disease processes, identify new therapeutic targets, or help select the most appropriate therapy for an individual patient
c. The information generated from a microarray is very useful in a clinical setting. For example, it can be used to predict risk of breast cancer recurrence and prescribe treatment accordingly; the oncotype Dx has resulted in a 30-40% decrease in the over treatment and under treatment of breast cancer.
Identify features of RNA pol II transcripts that direct their maturation
b. Changes in phosphorylation (usually at serine residues) of the RNA polymerase II C-terminal tail initiates processing by different proteins (for example, splicing proteins add the 5' cap)
Relate genetic features with clinical features of a DNA repeat expansion disease
d. The number of repeats in the DNA is related to the severity of the disease as well as the age of onset. Anticipation, or the progressive increase in disease severity and decrease in age of onset seen with successive generations in an affected family, is evident in trinucleotide expansion diseases.
Describe the reaction catalyze by ligase
i. DNA ligase synthesizes the phosphodiester bond that links Okazaki fragments. In the first step, AMP is attached to a 5' phosphate via the release of pyrophosphate (ATP is used). In the second step, the 3' hydroxyl group attacks the 5' phosphate, and AMP is released.
Function of miRNA
miRNA: microRNA, regulate gene expression by blocking translation of specific mRNA and cause their degradation
What is the pH of a solution when the concentration of an acid and its conjugate base are equal?
pH =pKa
Function of siRNA
siRNA: small interfering RNA, turn off gene expression by directing the degradation of selective mRNA and the establishment of compact chromatin structures
Function of snRNA
snRNA: small nuclear RNA, function in a variety of nuclear processes, including the splicing of pre-mRNA
snRNP
snRNP: the complexes that facilitate splicing
Function of snoRNA
snoRNA: small nucleolar RNA, helps to process and chemically modify rRNA
Function of tRNA
tRNA: transfer RNA, central to protein synthesis as adaptors between mRNA and amino acids
Describe the basic structure of a beta strand and indicate how the adjacent amino acid carbonyl oxygen atoms are pointed relative to each other
Adjacent amino acid carbonyl oxygen atoms point in opposite directions; every other amino acid carbonyl oxygen will hydrogen bond with the other strand of amino acids
Describe 5' to 3' polymerization in DNA replication
All DNA is polymerized in a 5' to 3' direction; this means that the DNA is read in the 3' to 5' direction.
Identify the general transcription factors and describe their functions
1. TFIID binds the promoter with TBP a. TBP (TATA binding protein) binds the TATA box and introduces a kink with marks the promoter as an active promoter (it widens the minor groove, allowing transcription factors and RNA polymerase II to get closer to the DNA) b. It there is no TATA box, TAFs interact with the promoter in a sequence-specific manner and force TBP to interact with the TATA box in a non-specific manner 2. TFIIA and TFIIB are recruited and bind the promoter 3. RNA polymerase II, TFIIE, and TFIIH are recruited a. RNA polymerase II binds the transcription start site b. TFIIE and TFIIH bind the promoter i. TFIIH has DNA helicase activity: it opens the DNA double helix only at the very beginning of transcription (ATP-dependent reaction); once RNA polymerase II begins synthesizing, the opening of the double helix becomes more favorable (no ATP required) ii. TFIIH has kinase activity: it phosphorylates the RNA polymerase II tail (C-terminal), which loosens its grip on the transcription start site and prompts it to begin transcribing (transcription factors dissociate, and other proteins are recruited in the process as well).
Indicate the average size of a domain
100 amino acids
Name complementary base pairs
A —> T (U in RNA); 2 hydrogen bonds C —> G; 3 hydrogen bonds
How much of genomic DNA is repetitive?
About half
How much of genomic DNA is unique?
About half
Explain why domains are referred to as building blocks or modules
Because they carry out the essential functions of a protein
Relate the oxidation state of the heme iron to oxygen binding
Ferrous (+2) iron can bind oxygen; ferric (+3) iron cannot bind oxygen.
Define a superfold
Folds that are frequently found in proteins; these are common because they are very stable
Understand the distinction between genetic and epigenetic regulation
Genetic gene regulation is accomplished via changes in the DNA sequence. Epigenetic gene regulation does not change the DNA sequence; rather, it changes other properties of the chromatin (for example, through covalent modifications of the histone tails). Epigenetic states can be inherited through various mitotic cell generations.
What double strand break repair mechanism is preferred in G0/G1?
NHEJ
Compare the plasma protein patterns of normal and multiple myeloma patients
Normal patients will have a spike of human serum albumin accompanied by low peaks of globulins. Multiple myeloma patients will still have a spike of human serum albumin, but there will be an abnormal spike among the low-peak globulins as well
Indicate whether or not all possible angles of phi and psi are possible
Not all phi and psi angles are possible due to steric hindrance
Define parallel and anti parallel beta sheets
Parallel = all of the strands run in the same direction (N to C) Anti parallel = the direction of the strands will alternate (N to C, C to N, N to C)
cDNA synthesis
Performed by reverse transcriptase, a polymerase found in retroviruses that catalyzes the formation of DNA from RNA
Aromatic amino acids
Phenylalanine, tyrosine, tryptophan
Oxygen saturation
The percent of myoglobin or hemoglobin that is bound to oxygen
Explain the role of primers and why they are RNA and not DNA; compare the synthesis and removal of primers
The primers are RNA because they automatically mark the sequence as suspect to be removed and replaced (contribute to high fidelity). RNA primers must be laid down so that DNA polymerase can begin synthesis in the 5' to 3' direction. The RNA primer is synthesized by DNA primase and removed by RNAseH.
Compare recognition mechanisms utilized by excision repair pathways
a. In BER, damage (alkylation, deamination, oxidation) is recognized by specific glycosylases (at least six types known). In NER, damage (non-specific bulky lesions) is recognized by XP proteins at any position in the DNA and at any point in the cell cycle. In TCR, damage is recognized by RNA polymerase during active transcription. In MMR, damage is recognized by MutS.
Meiotic prophase
This is when crossing over can occur (homologous chromosomes are lined up)
Prosthetic group
Tightly bound to the active site and regenerated after the chemical reaction (for example, heme)
Indicate the range of pO2s in tissues and the pO2 in lung capillaries
Tissues have a pO2 of about 20 torr; lung capillaries have a pO2 of about 90 torr
Indicate the main function of enzymes
To catalyze, or increase the rate of, chemical reactions
Describe the function of the active site
To directly participate in the making or breaking of bonds
Function of telomeres
To protect single-stranded chromosomes end - left by removal of primer RNA at the ends of linear replicated chromosomes -from recombination, fusion, and from being recognized as damaged DNA
Understand the use of DNA topoisomerase inhibitors in cancer treatment
j. DNA topoisomerase II inhibitors can be effective cancer treatments because they prevent the ligation of cleaved DNA molecules, resulting in the accumulation of single- or double-stranded DNA breaks, the inhibition of DNA replication and transcription, and apoptotic cell death.
Indicate why glycine occurs in the amino acid sequence every third residue in collagen
Glycine occurs in the amino acid sequence every third residue because it makes up the interior of the three-chain super helix. This allows the triple helix to be very tightly wound
Small amino acids
Glycine, alanine
Explain why HbF enhances delivery of oxygen to the fetus
HbF enhances delivery of oxygen to the fetus because it has a higher affinity for oxygen that adult hemoglobin.
Describe the quaternary structure of hemoglobin
Hemoglobin is a tetramer consisting of two identical alpha chains and two identical beta chains.
Explain why hemoglobin is much better at delivering oxygen to tissues than myoglobin would be
Hemoglobin is much better at delivering oxygen to tissues than myoglobin would be because the pO2 of oxygen in tissues is less than the P50 of hemoglobin. The P50 of myoglobin is much less than the pO2 of tissues, so myoglobin would not be able to easily let go of its oxygen in the tissues.
Indicate which conformational state the hemoglobin must be in for sickling to occur
Hemoglobin must be in the T-conformation (deoxygenated state) for sickling to occur.
Compare features of highly condensed and less condensed chromatin
Highly condensed chromatin is less active (genes are not expressed) and is found on the nuclear periphery, whereas less condensed chromatin is more active (it allows for access by proteins, which results in gene expression) and is found on the interior of the nucleus where transcription machinery is located.
Describe the methods of 2D electrophoresis and mass spectrometry, and how they could be used to identify protein bio markers for the diagnosis of cancers
In 2-D electrophoresis, proteins are separated by isoelectric point along the x-axis (or horizontal axis) and by size along the y-axis (or vertical axis). Separation of proteins by isoelectric point is achieved by isoelectric focusing, while separation of proteins by size is achieved by sodium dodecyl sulfate (SDS) gel electrophoresis. In mass spectrometry, a spot from the 2-D electrophoresis is cut out and digested with a protease (usually trypsin). The resulting fragments are ten separated by high performance liquid chromatography, which yields a peptide mass profile that can be compared to databases of known peptides. Although a cancerous tumor can be removed by current technology, 2-D electrophoresis and mass spectrometry would allow physicians to determine the peptides or proteins that are released by cancerous tumors and allowed to infect healthy cells in other parts of the body.
Compare DNA replication on leading and lagging strands
The leading strand is synthesized continuously in the direction of the replication fork, while the lagging strand is synthesized discontinuously in the direction opposite the replication fork. Okazaki fragments occur along the lagging strand, each of which is preceded by a RNA primer. DNA replication on the leading and lagging strand is facilitated by the same DNA polymerase; it is know as DNA POLE on the leading strand and DNA POLD on the lagging strand.
Indicate the main functions of myoglobin and hemoglobin
The main function of myoglobin is to store oxygen in skeletal muscle. The main function of hemoglobin to transport oxygen from the lungs to tissues
Describes important features of a replication origin and the mechanism for the fork initiation reaction
The replication origin is typically a sequence rich in adenine and thymine (since they form only two hydrogen bonds, the base pairing is easier to disrupt than a C-G pair). DNA replication is bidirectional (leading and lagging strand synthesis occurs simultaneously), and two forks move in opposite directions from one replication origin. Initiator proteins first bind to the replication origin and destabilize the AT-rich sequence. Then, DNA helicase is loaded onto the DNA strand via a helicase-loading protein. Once bound to the DNA strand, the helicase-loading protein jumps off, activating helicase. DNA primase is the next to jump on the strand to lay down the RNA primer, which enables DNA polymerases to start synthesizing the new chains.
Indicate the approximate number of genes and how their transcription can ultimately lead to >1 million proteins
There are approximately 20,000-25,000 genes, but their transcription can ultimately lead to more than one million proteins because of alternate promoters, alternate splicing, mRNA editing, and post-translational modifications, such as phosphorylation, methylation, and ubiquintylation
Consequences of unrepaired DNA damage
Transient cell cycle arrest, inhibition of cellular processes leading to apoptotic cell death or chromosome aberrations that eventually lead to excessive cell proliferation
Describe the principles behind Southern and Northern blotting and outline the steps involved in performing a Southern or Northern blotting experiment
a. A Southern blot beings with a restriction enzyme digest to create fragments of DNA that can be visualized on an agarose gel. The DNA fragments are then separated by size by agarose gel electrophoresis. Following electrophoresis, the gel is exposed to a high pH to make single-stranded DNA. The next step is to transfer the gel to a membrane; this is because the gel is too fragile to manipulate. This is accomplished by the following arrangement from top to bottom: book —> stack of paper towels —> membrane —> gel —> sponge —> buffer. As the salt buffer, travels upwards, it picks up the DNA fragments and carries them to the membrane. This creates an exact copy of the gel on the membrane. Then, a radio labeled probe is hybridized to the DNA fragments, and the labeled fragments are visualized by autoradiography. A Northern blot follows the same principles with a few exceptions: the sample is RNA; the buffer is different; and there is no treatment needed before the transfer because RNA is already single-stranded.
Distinguish between the types of small mutations, how they occur, and how they can affect gene regulation
a. A deletion mutation occurs when one or more nucleotides are eliminated from a sequence; it may (non-multiple of 3) or may not (multiple of 3) result in a frame shift mutation (all downstream amino acids are affected). An insertion mutation occurs when a copy or non-copy (rare in humans) transposition moves to a new location. It may (non-multiple of 3) or may not (multiple of 3) result in a frame shift mutation (all downstream amino acids are affected). An altered mutation refers to the replacement or modification of a single base; it is called a transition when a purine is changed to a purine or a pyrimidine is changed to a pyrimidine and a trans version when a purine is changed to a pyrimidine and vice versa. An altered base also includes methylation of bases and incorporation of unnatural bases. A missense mutation refers to an altered amino acid sequence (for example, a valine is changed to a lysine). A non-sense mutation results in early chain termination. A single nucleotide polymorphism (SNP) is a difference in a single base pair; an SNP may or may not (if it occurs at the wobble position) result in an missense mutation.
Explain what a dideoxynucleotide is and how it is used in standard sequencing experiments (chain terminator sequencing)
a. A dideoxyribonulceotide is simply a deoxyribonucleotide that is missing the hydroxyl group. Dideoxyribonucleotides can be used is standard sequencing experiments because they are chain terminators. Although a dideoxyribonucleotide can be incorporated into a new strand of DNA just as a deoxyribonucleotide would, it causes chain termination due to the missing reactive hydroxyl group. Dideoxyribonucleotides can be labeled with different fluorescent colors and take the place of deoxyribonucleotides in a PCR reaction to sequence DNA. The results can be visualized once the gel is passed through a laser and detector. Note that the results give you the sequence of the complementary strand.
Define the term palindrome
a. A palindrome is a DNA sequences that reads the same when it is flipped 180 degrees around its central axis. The two nucleotides that sandwich the central axis will base pair as will the adjacent pair as you move further from the central axis, and so on.
Describe how a specific DNA molecule can be introduced into a plasmid for its subsequence propagation in a bacterial cell
a. A specific DNA molecule can be introduced into a plasmid by cleaving the plasmid with a restriction enzyme. Once the plasmid is cleaved, the DNA fragment to be cloned is added to the plasmid through covalent linkage by DNA ligase.
Describe an example of direct repair
a. An example of direct repair involves the correction of an O6-meG base. O6-meG can base pair with C or T, and it blocks replicative polymerases. MGMT is the enzyme that converts O6-meG back into guanine that correctly base pairs with cytosine.
Identify common DNA-repeat expansion diseases
a. Common DNA repeat expansion diseases include progressive myoclonus epilepsy, fragile X syndrome, Friedreich ataxia, Huntington's disease, and myotonic dystrophy.
Describe factors that contribute to gene replication timing
a. Eukaryotic DNA replication only happens in the S phase of the cell cycle.
Describe the major steps of general recombination
a. First, the sister chromatids must line up after the double stranded break occurs. Then, a nuclease comes in and digests the 5' ends of the broken strands, creating 3' overhangs. The 3' overhang nearest the sister chromatid invades the sister chromatid with the help of RecA/Rad51 proteins (ATPase); this forms a triple-strand intermediate. Complementary base pairing allows repair polymerase to synthesize DNA using the undamaged DNA as a template. Lastly, the invading strand is released with the help of RubA/RubB proteins, and synthesis continues using the damaged DNA as a template. The nicks are sealed by DNA ligase.
Compare the features of a gene transcribed by RNA pol II, a pre-mRNA, and a mature mRNA
a. Gene transcribed by RNA polymerase II: this contains all of the features in the above diagram Pre-mRNA this contains only exons and introns (not really a thing because processing begins before transcription is finished - for example, the 5' cap is added after the first 20-30 nucleotides are incorporated) Mature mRNA: this contains only exons as well as a 5' cap and 3' polyA tail
Compare recombination outcomes in mitotic and meiotic cells
a. General recombination in meiosis is facilitated by specific enzymes. Spo11 initiates the double stranded break, and MRE11 uses its nuclease activity to remove Spo11 and chew back the 5' ends. Then, strand invasion begins with a RecA-like protein, and DNA polymerase synthesizes according to the undamaged strand. More than 90% of recombination events in meiosis will not result in a crossover. Somatic cell recombination is rare, but it can happen when homologous chromosomes line up during mitosis. There can be an exchange of genetic information that causes the genotypes of the daughter cells to be non-identical to the genotype of the parent cell (Aa—>AA and aa). This is referred to as loss of heterozygosity (LOH). Another mechanism for LOH is the invasion of the wrong sister chromatid following a double stranded break (Aa—>Aa and aa)
Describe the organization of pre-rRNA genes in the human genome and compare a pre-rRNA with a mature rRNA
a. Humans have 200 rRNA genes spread throughout the genome; if one promoter on one rRNA gene is defective, there will no overall effect. The 45S rRNA precursor undergoes extensive chemical modification facilitated by guide RNAs and cleavage that results in 18S rRNA, 5.8S rRNA, and 28S rRNA.
Describe significant features of DNA-repeat expansion diseases
b. Progressive myoclonus epilepsy, fragile X syndrome, and Friedreich ataxia inhibit transcription, whereas Huntington's disease results in the accumulation of a non-functional protein.
Explain how a DNA sequence can be used to predict the amino acid sequence of a protein that it encodes
b. A DNA sequence can be used to predict the amino acids sequence of a protein that it encodes by finding the start codon and translating subsequent triplets (codons) into the universal genetic code until you reach a stop codon.
Explain the term restriction fragment length polymorphism (RFLP) when comparing DNA from different individuals
b. A restriction fragment length polymorphism (RFLP) occurs when digestion with the same restriction enzyme results in DNA fragments of different lengths for different individuals (for example, digestion by MstII in HbA v. HbS).
Recognize the different types of large scale mutations that can lead to chromosome deletions, translocations, duplications, and inversions
b. An application/duplication results in three copies of the duplicated region. A deletion at the chromosomal level can remove hundreds to thousands of genes, resulting in just one copy of the deleted region. Translocations (inversions and fusions/insertions) may affect gene expression if they interrupt a coding region of DNA.
Compare molecular end clinical features of the 3 classes of repair mechanisms
b. Direct repair: damage reversal occurs on the base (base in NOT removed) DNA methyl transferase (MGMT): increased activity may protect against chemically induced tumor formation but render tumors resistant to chemotherapires; decreased activity may predispose for chemically-induced brain tumors Excision repair (BER, NER/TCR, MMR): removal and replacement of damaged base BER: glycosylase deficiencies may predispose for human disease (cancer) NER: mutations in NER genes result in XP and other related diseases with varying degrees of cancer susceptibility MMR: mutations in MMR genes associated with 90% of colorectal cancer Double-strand break repair: homologous end joining (HEJ) and non-homologous end joining (NHEJ) HEJ: rare consequence can be loss of heterozygosity leading to tumorigenesis NHEJ: highly mutagenic; decreased activity may result in immunodeficiency syndrome
Explain how cDNA is prepared and when cDNA would be used in PCR
b. First, mRNA is purified hybridized with a poly-T primer (binds to the poly-A tail — mRNA is the only RNA that has poly-A tail). Then, you make cDNA by adding reverse transcriptase (you have DNA/RNA hybrid at this point). Next, you degrade the RNA with RNAseH and synthesize a second cDNA strand using DNA polymerase (the RNA fragment acts as a primer). You left with a double-stranded cDNA copy of the original mRNA. cDNA would be used in PCR instead of DNA to analyze an intact gene.
Describe an approach for using gene therapy to treat cystic fibrosis
b. Gene therapy could be used to treat cystic fibrosis by introducing the affected individual to a functional CFTR protein. This could be accomplished by creating a plasmid containing the CFTR gene using recombinant DNA technology, packaging the plasmid in a viral vector, and delivering the virus to affected individuals via aerosolization. The viral vector would be incorporated into the host genome and transcribed to produce a functional ion channel.
Explain how de novo mutations arise
b. Germline mutations arise in the gamete of an individual, which if fertilized will result an affected individual; the affected individual can then pass on the mutation to his or her offspring. De novo mutations can arise from copying errors, spontaneous chemical attack (depurination/deamination), and environmental exposure to natural radiation and reactive metabolites.
Identify a chromatin property that would contribute to early v. Late replicating DNA
b. Heterochromatin is replicated later compared to euchromatin. Gene-dense regions replicate earlier than gene-poor regions. Regions with highly expressed genes in a given tissue replication earlier than gene regions that are not expressed or expressed at a lower level in that tissue (perhaps due to the opening of chromatin in those regions).
Explain the clinical significance of LOH
b. Loss of heterozygosity is clinically significant because it can result in disease or a change in phenotype.
Describe how a microarray experiment would be performed
b. Microarray experiment would be performed by collecting and isolating mRNA molecules, creating single-stranded cDNA molecules labeled with fluorescence, and pipetting the cDNA onto a DNA microarray, a slide on which copies of single-stranded DNA fragments (complementary to mRNA of gene of interest) are fixed. Complementary cDNA sequences hybridized with the fixed DNA fragments and the unbound cDNA is washed off. The relative amounts of each cDNA is quantified through the intensity of the fluorescence.
Speculate as to why DNA damaging agents might be clinically useful
b. On the contrary, O6-meG inducing drugs can be useful as anti-cancer drugs because they overwhelm the cell with damage, causing the cell to go into apoptosis.
Describe how PCR can be used to diagnose a specific genetic disease such as hemophilia 09o
b. PCR exponentially amplifies a PCR product because two copies are made for every piece of double-stranded DNA at the start of a cycle. If you start with one double-stranded DNA molecule, you will have two at the end of cycle 1, four at the end of cycle 2, eight at the end of cycle 3, and so on.4c. In the case of hemophilia, PCR can be used to amplify the factor VIII gene. The PCR product can then be cut with the BclI restriction enzyme and visualized through agarose gel electrophoresis. Since the BclI cut site is mutated in hemophilia, BclI will not cut the PCR product from an affected individual, yielding one fragment on the gel; it will cut the PCR product from an unaffected individual, yielding two fragments on the gel and allowing for diagnosis.
Explain how NGS and whole genome sequencing might be useful in clinical medicine
c. A focused NGS panel may be used in a clinical setting to compare an individual's genome to 23-150 known mutations of cystic fibrosis. Whole genome sequencing, although not yet routine in clinical practice, could be used to identify any mutations in an individual's genome to diagnosis disease.
Describe the fate of nucleosomes during DNA replication
c. H2A-H2B dimers are removed from the template strand prior to replication, while the H3-H4 tetramer tends to remain loosely bound to the template strand throughout replication. Once replication is finished, NAP1 reloads H2A-H2B dimers onto the newly synthesized strand, and CAF1 reloads and H3-H4 tetramer that were removed during the replication process. The H3-H4 tetramer as that remained bound to the template strand serve as bookmarks to re-establish histone marking patterns.
Describe the molecular techniques used to examine a potential RFLP
c. Molecular techniques used to examine a potential RFLP include digestion by restriction enzyme —> agarose gel electrophoresis —> hybridization —> Southern blot —> probe.
Relate the location of the DNA expansion with effect on transcription and translation
c. Promoter (progressive myoclonus epilepsy) = inhibits transcription 5' untranslated region (fragile X syndrome) = inhibits transcription Intron (Friedreich ataxia) = inhibits transcription Open reading frame = non-functional protein
Explain the functions of snRNP molecules in the maturation of mRNA
c. The snRNPs are composed of an snRNA with at least 7 proteins; these make up the spliceosome and are responsible for catalyzing the splicing reaction. Cleavage and polyadenylation specificity factor (CPSF) and cleavage stimulation factor (CSTF) travel with the RNA polymerase II C-terminal tail and initiate cleavage and polyadenylation of the mRNA following recognition of specific sequences on the mRNA. CPSF recognizes AAUUAA prior to the transcription termination site, and CSTF recognizes a GU-rich or U-rich region after the transcription termination site. Once the cleavage is complete, polyA polymerases (PAP) adds adenines to the 3' end without the use of a template, and polyA binding proteins bind the polyA tail once completed.
Determine the relative length of a DNA fragment from its electrophoretic migration
c. To determine the relative length of a DNA fragment from its electrophoretic migration, compare it to the DNA size markers (standard).
Explore the potential of a telomerase template antagonist as an anti cancer agent
d. A telomerase template antagonist could be used as an anti cancer agent because it would promote the loss of chromosome ends in cancer cells, eventually resulting in the loss of essential genes and apoptotic cell death.
Describe how DNA fragments migrate in gel electrophoresis based on their relative charge
d. DNA is negatively charged (due to the phosphate backbone), so it will travel towards the anode (positive electrode) when it is placed near the cathode (negative electrode).
Explain the basics of the CRISPR/Cas9 system for gene editing
d. The CRISPR/Cas9 system is a gene editing system that allows for precise changes to be made in DNA. This is a naturally occurring system in bacteria that serves to destroy foreign DNA. The system is made up of the Cas9 enzyme, a guide RNA, and target specific portion of the guide RNA. Cas9 scans the DNA until it finds a sequence that is complementary to the target specific portion of the guide RNA (note that the sequence must by followed by a PAM). Then, it will cause a double-double stranded break in the DNA, allowing for gene disruption, correction, and insertion.
Compare the function and mechanisms of helicase and topoisomerases
f. Helicase is an allosteric motor protein that unwinds DNA at a rate of about 1,000 nucleotide base pairs per second in an ATP-driven reaction. DNA topoisomerase I relieves supercoiling ahead of the replication fork (few hundred to several kilo bases ahead); it does this by covalently attaching to a DNA phosphate via a tyrosine in its active site, breaking the phosphodiester bond in one strand of DNA. This allows the two ends of the DNA double helix to rotate relative to one another, relieving torsional strain. Spontaneous re-formation of the phosphodiester bond regenerates the DNA helix and the DNA topoisomerase. DNA topoisomerase II untangles DNA after it is replicated; it does this by creating a double-strand break in one strand and forming a protein gate, which allows the second DNA helix to pass through.
Describe fluorescent in situ hybridization (FISH) and discuss how it can be used in the diagnosis of disease
f. In Fluorescent In Situ Hybridization (FISH), a labeled probe (appears as different colors) binds to complementary sequence on a chromosome. It allows recognition of multiple target sequences simultaneously on a a chromosome or in a whole cell. FISH can be used in the diagnosis of Her2+ breast cancer in which a tyrosine kinase receptor is over expressed, for example.
Explain mRNA capping and its possible functions
f. The 7-methylguanine cap is attached to the 5' end of the pre-mRNA through a 5' to 5' linkage that is facilitated by a phosphatase, guanyl transferase, and methyl transferase. The purpose of the 5' cap is to identify the product as an mRNA, maintain its stability and protect it against degradation, and allow it to leave the nucleus.
Compare the role of SSB proteins in denature group and stabilizing DNA
g. Single-strand binding proteins (SSB) prevent re-annealing of the template strand prior to the arrival DNA polymerase. This serves to the prevent the formation of "hairpins," which will stop DNA polymerase from synthesizing. The SSBs are monomers that assemble cooperatively. Each SSB is composed of two domains that cover eight bases.
Describe the functions of the subunits of the replication machine and explain how leading and lagging strand synthesis occurs simultaneously
h. The replication machine refers to the idea that the proteins involved in DNA replication do not act independently; rather, they form one large multi enzyme complex that requires the lagging strand to fold back (see slide 22).
Function of rRNA
rRNA: ribosomal RNA, forms the basic structure of the ribosome and catalyze protein synthesis
