AAMC Content Guidelines UPDATED PDF

Credits to: mcat-review.org Khan Academy Examkrackers Kaplan r/MCAT A redditor, whose name we cannot find, who posted a sim...

Credits to: mcat-review.org Khan Academy Examkrackers Kaplan r/MCAT A redditor, whose name we cannot find, who posted a similar guide long ago Content Category 1A: Structure and function of proteins and their constituent amino acids Amino Acids (BC, OC) Description o Absolute configuration at the α position § The alpha carbon IN EVERY amino acid is a chiral center EXCEPT in glycine (it is achiral, since the R group is an H) § EVERY AA has S configuration EXCEPT FOR cysteine (R configuration) o Amino acids as dipolar ions § At low pH, amino acid = cationic § At high pH, amino acid = anionic § At pH = pI, amino acid = zwitterionic (neutral) o Classifications § Acidic or Basic ACIDIC: Aspartic Acid (Asp, D) ; Glutamic Acid (Glu, E) BASIC: Lysine (Lys, K) ; Arginine (Arg, K) ; Histidine (His, H) § Hydrophobic or Hydrophilic HYDROPHILIC: If the R group contains acids, bases, amines or alcohols o Arginine (Arg, R), Lysine (Lys, K), Aspartic Acid (Asp, D), Glutamic Acid (Glu, E), Glutamine (Gln, Q), Asparagine (Asn, N), Histidine (His, H), Serine (Ser, S), Threonine (Thr, T), Tyrosine (Tyr, Y), Cysteine (Cys, C), Tryptophan (Trp, W) HYDROPHOBIC: If the R group DOES NOT contain what is listed above ^^ o Alanine (Ala, A), Isoleucine (Ile, I), Leucine (Leu, L), Methionine (Met, M), Phenylalanine (Phe, F), Valine (Val, V), Proline (Pro, P), Glycine (Gly, G) Reactions o Sulfur linkage for cysteine and cystine § Cysteine = amino acid with the thiol R group § Cystine = 2 cysteines that have formed a disulfide bond o Peptide linkage: polypeptides and proteins § Peptide bonds link amino acid chains together § Peptide bonds are formed by the nucleophilic addition-elimination (condensation, dehydration rxn) reaction between the carboxyl group of one amino acid and the amino group of another amino acid § The nucleophilic amino group attacking an electrophilic carbonyl § The bond when formed has a lot of resonance delocalization (partial double bond character all over the place!) Makes the bond very rigid/planar However, this is still free rotation around the ALPHA CARBON o Hydrolysis § The process of breaking the peptide bond Done by either acid/base hydrolysis (nonspecific) or with the help of proteolytic enzymes (specific) Protein Structure (BIO, BC, OC) Structure o 1o structure of proteins § Linear sequence of amino acids § Determined by the peptide bond linking each amino acid § Covalent (Peptide) bonds o 2o structure of proteins § Local structure, stabilized by hydrogen bonding α-helices – hydrogen bonds run up and down, stabilizing the structure β-pleated sheets – stabilized by hydrogen bonds connecting the sheets o Antiparallel vs. Parallel configurations § The way the linear sequence folds on itself § Determined by the backbone interactions (primarily hydrogen bonds) § Hydrogen bonds between backbone atoms o 3o structure of proteins § 3-D structure stabilized by hydrophobic interactions, acid-base interactions (salt bridges), hydrogen bonding, and disulfide bonds § Depends on distant group interaction Stabilized by hydrogen bonds, van der Waals, hydrophobic packing, disulfide bridge formation § Disulfide bond formation happens on the exterior of the cell (covalent bond of two cysteines) Extracellular space prefers the formation of disulfide bonds (the oxidizing environment) § Hydrophobic interactions and polar interactions between side chains o 4o structure of proteins § Interactions between subunits (multiple polypeptides) § Hydrophobic interactions and ionic bonds between side chains (i.e. cysteine side chains making disulfide bonds) Conformational stability o Denaturing and Folding § Primary Structure = determined by peptide bonds § Secondary Structure = determined by backbone interactions (hydrogen bonds) § Tertiary Structure = determined by distant interactions between groups (van der Waals, hydrophobic packing, disulfide, hydrogen bonding) § Quaternary Structure = determined by same bonds from tertiary structure § Protein is ONLY FUNCTIONAL when in the proper conformation A force that helps stabilize the protein is the solvation shell o Solvation shell = layer of solvent surrounding the protein (can be the water solvent interaction with polar AAs, etc.) § Denaturation = when a protein loses active conformation and becomes inactive Occurs by changing pH, temp, chemicals or even enzymes § If you denature by heating, you destroy all the structures of the protein except the primary structure (primary structure is conserved) o Hydrophobic Interactions/Solvation Layer (entropy) (BC) § The hydrophobic regions of the protein aggregate, which releases the water from cages à This increases the entropy of water, which is the major thermodynamically favorable component of protein folding Separation techniques o Isoelectric point  § pI is determined by averaging the pKa values that refer to the protonation and deprotonation of the zwitterion § Isoelectric focusing = gel electrophoresis method that separates proteins on basis of their relative contents of acidic and basic residues (gel with pH gradient is used) o Electrophoresis § Positively charged anode at bottom, negatively charged cathode at top § Larger molecules will have harder time moving, thus separation created by size with the smallest molecules towards the bottom § Native Page = retains structure of protein ; SDS-Page = break into subunits Non-Enzymatic Protein Function (BIO, BC) Binding (BC) o Bind various biomolecules – bind specifically and tightly o Receptors/Ion channels in the membrane: § Receptors bind or receive signaling molecules (ligand) which makes a chemical response (i.e. insulin receptor) § Ion channels can allow ions to enter/exit the cell Immune System o Antibodies = protein components of the adaptive immune system whose main function is to find foreign antigens and target them for destruction o Antigen = the ligand for antibodies § Antigens can be thought of as little red flags for the immune system letting us know, “Hey, that’s not supposed to be there!” Motors o Transport: e.g. Hemoglobin (at high concentration of ligand, have high affinity, at low concentration of ligand, have low affinity) o Myosin/Kinesin/Dynein § Myosin = responsible for forces exerted by contracting muscles § Kinesin/Dynein = motor proteins responsible for intracellular transport Dynein = plays a role in the motility of cilia Enzyme Structure and Function (BIO, BC) Function of enzymes in catalyzing biological reactions o Enzymes function to lower the activation energy of reactions (do not get used up!) o Structure determines function à change in structure = change in function Enzyme classification by reaction type o 6 Types of Enzymes: § Transferase Move a functional group from one molecule to another A + BX à AX + B § Ligase Join two large biomolecules, often of the same type A + B à AB § Oxidoreductase Catalyze oxidation-reduction reactions that involve the transfer of electrons Oxidase = oxidizing or taking away electrons from a molecule Reductase = reducing or giving electrons to a molecule A + B: ßà A: + B § Isomerase Interconversion of isomers, including both constitutional and stereoisomers AàB § Hydrolase  Cleavage with the addition of water A + H2 O à B + C § Lyase Cleave without the addition of water and without the transfer of electrons (reverse reaction, synthesis, is usually more biologically important) A à B + C (does not use water, or oxidation/reduction) Lyases generate either a double bond or a ring structure Reduction of activation energy o Acid/Base catalysis = enzymes use acidic/basic properties to make rxns go faster by proton transfer o Covalent catalysis = enzymes covalently bind to help with electron transfer o Electrostatic catalysis = charged molecules or metal ions used to stabilize big positive or negative charges o Proximity/Orientation effects = enzymes make collisions between reacting molecules happen more often o Transition state = highest energy point from path A to B (in Aà B) § Where you also find most instability (high energy = more unstable) o Enzymes lower the activation energy of the reaction (making it easier for the reactants to transition to form products) Substrates and Enzyme Specificity o Enzyme-substrate specificity derives from structural interactions o Enzymes can be specific enough to determine between stereoisomers Active Site Model o Location on the enzyme where it reacts with its substrate o Shape/characteristics (functional groups) of an active site are responsible for the specificity of the enzyme Induced-fit Model o Initial Binding = when the substrate first binds to the enzyme (not perfect) § Forces holding the two together are strong, but not at the maximum strength yet o Enzyme and substrate thus mold their shape to bind together super tightly § Called the induced fit because both the enzyme and substrate have changed their shape a little so they bind together really tightly (catalyzing reaction at full force) o Binding between reactant and enzyme STRONGEST at the transition state Mechanism of catalysis o Cofactors § Directly involved in the enzyme’s catalytic mechanism (might be stabilizing the substrates, or helping the reaction to convert substrates from one form to another) (e.g. Mg2+) o Coenzymes § Organic carrier molecules (i.e. NADH, CoA) o Water-soluble vitamins § Need to obtain from the diet § Vitamins à organic cofactors and coenzymes § e.g. Vitamin B3 is precursor for NAD § e.g. Vitamin B5 is precursor for CoA Effects of local conditions on enzyme activity o Enzymes work best in specific environments o Effects of pH changes: § e.g. DNA à Negatively charged à DNA Polymerase binds Mg2+ cofactor to stabilize negative charge on DNA  In normal conditions, DNA Pol holds onto Mg ion through electrostatic interaction between magnesium and one of its aspartate residues, which would be deprotonated and thus negatively charged at neutral pH values If you took DNA Pol and put it in environment with reduced pH, the aspartate residue would become protonated since pH has dropped so much, and protonated form has no negative charge, so can’t bind Mg ion cofactor DNA Pol cannot do job properly in low pH environment o Effects of temperature changes: § Proteins fold from primary à secondary à tertiary à quaternary structures to function properly § Significant changes in temp cause protein to lose its functionality (loses its shape) e.g. when we get sick and our body temperature goes up, our digestive enzymes cannot work properly and consequently we cannot eat food as well Control of Enzyme Activity (BIO, BC) Kinetics o General (catalysis) § Enzymes lower the activation energy of a reaction, or the ΔG of the transition state (NOT OF THE RXN!) § E + S ßà ES ßà E + P § At really high [S] the enzymes will be saturated Even if you increase concentration of [S] from this point, there will still be a Vmax o Michaelis-Menten § Vmax is defined for a specific enzyme concentration (adding more enzyme will increase the Vmax) § Michaelis-Menten equation calculates the rate of reaction using Vmax, the substrate concentration [S], and the Michaelis constant Km. Km = the [S] required to reach 1/2Vmax. § Km does not fluctuate with changes in [enzyme] and is indicative of enzyme-substrate affinity § Enzymes with high enzyme-substrate affinity will reach 1/2Vmax at a lower substrate concentration (Lower Km) § Lower enzyme-substrate affinities will result in needing a higher substrate concentration to reach 1/2Vmax (Higher Km) !"#$ ['] § V = )"*['] As substrate concentration increases, the reaction rate also increases until a maximum value is reached At ½ Vmax, [S] = Km § kcat = Vmax / [E]T = Enzyme’s “Turnover Number” How many substrates can this enzyme turn into product in one second at its maximum speed § Catalytic Efficiency = kcat / Km o Cooperativity § Some proteins can bind more than 1 substrate § Cooperativity = substrate binding changes substrate affinity § Positive Cooperative Binding = Substrate binding increases affinity for subsequent substrate § Negative Cooperative Binding = Substrate binding decreases affinity for subsequent substrate § Non-Cooperative Binding = Substrate binding does not affect affinity for subsequent substrate § TOW RIGH (Hemoglobin affinity for O2)  T state = Low affinity R state = High affinity o Feedback Regulation § When product of reaction binds allosteric site of the enzyme, affecting the catalytic activity Can be positive = increases enzyme-substrate affinity Can be inhibitory = reducing activity at the active site or inactivating it completely o Inhibition – Types § Competitive E (inhibitor binds to E here to make EI) + S ßà ES ßà E + P Blocks the enzyme and makes it unable to react with substrate to form product Inhibitor competes with substrate for space on the enzyme Binds: Active Site Impact on Km: Increases Impact on Vmax: No Change § Uncompetitive E + S ßà ES (inhibitor binds to the ES here to make ESI) ßà E + P Molecule that binds only to the enzyme-substrate complex, rendering it catalytically inactive Binds: Allosteric Site Impact on Km: Decreases Impact on Vmax: Decreases § Non-competitive Prevents the enzyme from turning substrate into product Binds to an allosteric site on the enzyme, causing a conformational change that decreases catalytic activity at the active site regardless of whether a substrate is already bound Binds: Allosteric Site Impact on Km: No Change Impact on Vmax: Decreases § Mixed Molecule that binds to an allosteric site on the enzyme, causing a conformational change that decreases catalytic activity at the active site Generally, have preference towards binding either the enzyme-substrate complex, or binding the enzyme alone Binds: Allosteric Site Impact on Km: Increase (if prefer enzyme w/o substrate) or Decrease (if prefer enzyme with substrate bound) Impact on Vmax: Decreases o Regulatory Enzymes § Allosteric Enzymes Allosteric site present, molecule binds it, can either upregulate or downregulate the enzyme function § Covalently-modified enzymes  Not all enzymes are proteins (i.e. Inorganic metals, small organic molecules like Flavin) Small Posttranslational Modifications: o Translation à synthesis of AA polymer o “Post-translation” à after initial synthesis o “Small” à adding or removing small functional groups Methylation o Modification of a protein that involves addition of methyl group (CH3) Acetylation o Modification of a protein that involves addition of an acetyl group Glycosylation o Addition of a sugar to a protein I.e. Acetylation of lysine residue on a protein o Electron withdrawing impact of the acetyl group will prevent nitrogen from carrying positive charge and modify the behavior of the amino acid Suicide Inhibition o Suicide inhibitors covalently bind the enzyme and prevent it from catalyzing reactions o Rarely unbind – why it’s called suicide (enzyme won’t work anymore) § Zymogens Inactive form of an enzyme that requires covalent modification to become active I.e. Digestive enzymes of the pancreas o Pancreas releases trypsinogen (a zymogen) o Once in the intestine, it is covalently modified by an enzyme called enterokinase to the active form Trypsin o This makes sure trypsin does not break down proteins that we need in the pancreas Content Category 1B: Transmission of genetic information from the gene to the protein Nucleic Acid Structure and Function (BIO, BC) Description o Nucleic acids can be DNA or RNA, single-stranded or double-stranded o Protein coat covers the nucleic acid o The 2 single-strands are anti-parallel to each other. Going from 5’ to 3’ of one strand means going 3’ to 5’ of other strand. Nucleotides and nucleosides o Nucleotide = base (Adenine, Guanine, Thymine, Cytosine) + sugar + phosphate o Nucleosides = base + sugar = Adenosine, Guanosine, Thymidine, Cytidine o Sugar phosphate backbone § Important structural component of DNA which consists of the pentose sugar and phosphate groups § Sugars linked together by a phosphodiester bond o Pyrimidine, purine residues § Adenine and Guanine are purines (2 rings) § Cytosine, Threonine, and Uracil are pyrimidines (1 ring) Base pairing specificity: A with T, G with C o A forms 2 hydrogen bonds with T o G forms 3 hydrogen bonds with C o GC bonds are stronger. DNA with high GC content harder to break apart. o Complementary strands of DNA hydrogen bond with each other. Function in transmission of genetic information o Because of the complementary nature of base pairing, DNA can transmit genetic information through replication DNA denaturation, reannealing, hybridization o Disruption of the hydrogen bonds, such as with high temperature, can cause the unwinding of the two strands (denaturation), which can then also be brought back together when proper conditions return (reannealing) o A single strand of DNA will readily bind another single strand DNA in process of hybridization where there is significant amount of base pair matching between their sequences DNA Replication (BIO) Mechanism of replication: separation of strands, specific coupling of free nucleic acids o 1. Double-stranded DNA must separate or unwind. To do this: § DNA gyrase (class II topoisomerase) responsible for uncoiling the DNA ahead of the replication fork § Helicase responsible for unwinding DNA at replication fork § Single-strand binding protein (SSB) responsible for keeping DNA unwound after helicase. SSBs stabilize ssDNA by binding to it. o 2. You start making DNA that is complementary to the unwound/separated DNA. Note, all biological DNA synthesis occurs from 5’ to 3’ end. § Primase lays down short RNA primer on unwound DNA. Primer made of RNA but is complementary to DNA sequence. Later, this RNA is replaced with DNA. § DNA polymerase takes over and makes DNA that is complementary to unwound DNA. § DNA synthesis occurs on both strands of unwound DNA. Synthesis that proceeds in direction of replication fork is leading strand. Synthesis that proceeds in opposite direction to replication fork is lagging strand. Lagging strand contains Okazaki fragments. o 3. RNA primers replaced with DNA by a special DNA polymerase. Okazaki fragments in lagging strands are stitched together by DNA ligase. o DNA synthesis is bidirectional: 2 replication forks form and proceeds in opposite directions.  o Biological DNA synthesis always proceeds from 5’ to 3’ end. o DNA polymerase has proofreading activity, corrects any mistakes (mutations) it makes o Replication occurs once every cell generation, during the S phase. (Cell division may occur twice in meiosis, but replication still only occurs once) Semi-conservative nature of replication o Newly synthesized DNA contains one old strand and one new strand o Meselson and Stahl proved this by experiment: used heavy (15N) DNA as old (pre-replication) DNA and used light (14N) nucleotides for synthesis of new DNA. They can tell difference between heavy and light DNA by centrifugation. They found that when heavy DNA undergoes one round of replication in light nucleotides, the DNA is made of intermediate weight. After second round of replication, DNA is split between intermediate and light weight. o If DNA replication were completely conservative, only heavy and light DNA would be seen, nothing in between. o If DNA replication were dispersive, everything would be of intermediate weight. This was not the case because after second round of replication, light DNA was seen. Specific enzymes involved in replication o Helicase = uses hydrolysis of ATP to “unzip” or unwind DNA helix at replication fork to allow resulting single strands to be copied o Primase = polymerizes nucleotide triphosphates in a 5’ to 3’ direction. Synthesizes RNA primers to act as a template for future Okazaki fragments to build on to. o DNA Polymerase III = synthesizes nucleotides onto leading end in classic 5’ to 3’ direction. o DNA Polymerase I = synthesizes nucleotides onto primers on lagging strand, forming Okazaki fragments. This enzyme cannot completely synthesize all the nucleotides. o Ligase = glues together Okazaki fragments, an area DNA Pol I unable to synthesize o Telomerase = catalyzes lengthening of telomeres; enzyme includes molecule of RNA that serves as template for new telomere segments o Nuclease = excises or cuts out unwanted or defective segments of nucleotides in DNA sequence o Topoisomerase = introduced single-strand nick in the DNA, enabling it to swivel and thereby relieve the accumulated winding strain generated during unwinding of double helix o Single Strand Binding Proteins = holds the replication fork of DNA open while polymerases read the templates and prepare for synthesis Origins of replication, multiple origins in eukaryotes o Process of DNA replication begins at origin of replication, where molecule’s two strands are separated, producing a replication bubble with two replication forks unzipping the DNA bidirectionally away from the origin. o Prokaryotes have single origin of replication for their single, circular DNA o Eukaryotes have multiple origins of replication across their numerous linear chromosomes Replicating the ends of DNA molecules o Linear chromosomes arrive at issue with replication at ends of their lagging strands by which a portion of the strand at the very end (located in telomere, a region of repetitive sequences at the end of the chromosome) is unable to by synthesized due to lack of 3’ end of a nucleotide to extend from o This results in shortening of telomeres in linear chromosomes after numerous rounds of replication o Issue resolved in presence of telomerase which lengthens telomeres with repetitive sequences, thus protecting them from loss during replication Repair of DNA (BIO) Repair during replication o DNA polymerase has proofreading activity (also called 3’ à 5’ exonuclease activity). If a wrong nucleotide gets incorporated, polymerase will “back-up” and replace it with correct one  o Special polymerase that replaces the RNA primers with DNA also have 5’ à 3’ activity. This allows polymerase to clear away short stretches of incorrect nucleotides (RNA or incorrect DNA) and replace it with the right ones (DNA). Repair of mutations o Mismatch repair: enzymes recognize incorrectly paired base-pairs and cuts out stretch of DNA containing the mismatch. Then polymerase re-adds the correct nucleotides in. § During mismatch repair, repair enzyme must decide what strand of DNA to cut since DNA contains 2 strands. To do this, the enzyme cuts DNA strand that does not have methylations. The original (old) DNA has methylations but newly synthesized DNA does not have them until shortly after replication. Thus, there is a short period when mismatch repair enzymes can find out what strand to cut if mismatch is encountered. o Base-excision repair: a damaged base gets cut out. Then the base’s sugar phosphate backbone gets cut out. Several more nucleotides next to base get cut out. Finally, polymerase remakes the cut-out nucleotides. o Nucleotide-excision repair: damaged nucleotide(s) get cut out then polymerase replaces it. This is like mismatch-repair, but not for mismatch. It’s for damages like thymine dimers, and other damages that changes normal nucleotides into abnormal nucleotides. o Nick translation: basically 5’ à 3’ exonuclease activity coupled to polymerase activity. The polymerase chugs along, chews off bad nucleotides and replaces them with new nucleotides. This is what happens when RNA primers are replaced with DNA. o SOS response in E. Coli: during replication, when there’s too much DNA damage for normal repair to handle, the SOS repair system comes along. Instead of correcting any DNA damages during replication, polymerase replicates over the damaged DNA as if it were normal. By using damaged DNA as template error rates are high, but still better than not replicated at all. Genetic Code (BIO) Central Dogma: DNA à RNA à protein o DNA: Resides in nucleus. Codes information in genes. o Transcription: Inside the nucleus, the DNA genes get transcribed into RNA (messenger RNAs or mRNAs) o RNA: The mRNAs get transported out of nucleus into cytoplasm. mRNAs are working copies of the gene. o Translation: Ribosomes read off mRNAs to make proteins. o Protein: Synthesized by ribosomes. End product of what’s encoded in the genes and they perform all functions in the cell. The triplet code o Codon: The mRNA is a sequence of nucleotides, but it codes for a sequence of amino acids. To do this, every 3 nucleotide codes for an amino acid. These triplets of nucleotides are called codons. A single mRNA contains many codons. § Codons are continuous, non-overlapping and degenerate. § Continuous because one codon follows right after another. There are no nucleotides in between. § Non-overlapping because the 3 nucleotides that consist one codon never serve as part of another codon § Degenerate (see below) o Anticodon: the 3 bases on the “tip” of the tRNA. A single tRNA contains a single anticodon at the “tip” and the corresponding amino acid at the “tail.” Anticodons are complementary to their corresponding codon. Codon-anticodon relationship o During translation, codons pair with anticodons so that the correct amino acids can be linked to a given codon. Degenerate code, wobble pairing o Genetic code is degenerate because more than one codon codes for a given amino acid  o We refer to variable third base in the codon as the wobble position. Wobble is an evolutionary development designed to protect against mutations in the coding regions of our DNA. Mutations in the wobble position tend to be called silent or degenerate, which means no effect on the expression of the amino acid and therefore no adverse effects on the polypeptide sequence Missense, nonsense codons o Missense codon: mutated codon that results in a different amino acid o Nonsense codon: mutated codon that results in a stop codon Initiation, termination codons o Initiation codon (AUG): signals the start of translation. Lies just downstream of the Shine Dalgarno sequence (Kozak sequence for eukaryotes) o Termination codon (UAG, UGA, UAA): signals the end of translation. Unlike other codons, tRNA are not involved. A protein called “release factor” comes along and terminates translation. Messenger RNA (mRNA) o mRNA stands for messenger RNA. It’s the product of transcription and the template for translation o The 5’ cap is a modified nucleotide linked in a special way to mRNA. This protects 5’ end from exonuclease degradation. o The poly-A tail protects 3’ end of mRNA from exonuclease degradation o Eukaryotic mRNA: 5’ cap - nucleotides - 3’ poly-A tail o Prokaryotic mRNAs don’t have 5’ cap or poly-A tail Transcription (BIO) Transfer RNA (tRNA); ribosomal RNA (rRNA) o Both tRNA and rRNA are products of transcription. However, they do not serve as template for translation. tRNA responsible for bringing in correct amino acid during translation. rRNA makes up ribosome, enzyme responsible for translation. o tRNA made of nucleotides, many of which are modified for structural and functional reasons. At the 3’ end, the amino acid is attached to the 3’OH via an ester linkage. o tRNA structure: clover leaf structure with anticodon at tip, and amino acid at 3’ tail. o rRNA made of nucleotides, many modified for structural and functional reasons o rRNA highly structured because it contains active site for catalysis. The rRNA of large ribosomal subunit is responsible for catalyzing peptide bond formation and can do this even without ribosomal proteins. Mechanism of transcription (RNA polymerase, promoters, primer not required) o 1. Chain Initiation: RNA polymerase binds to promoter (TATA box) of dsDNA (closed complex). Double stranded DNA template opens up (open complex) o 2. Chain Elongation: nucleoside triphosphates (AUGCs) adds corresponding to the DNA template. No primer is required. RNA elongates as RNA polymerase moves down DNA template. RNA made from 5’ to 3’ direction. o 3. Chain Termination: 2 ways that transcription can terminate: § 1. Intrinsic termination: specific sequences called a termination site creates a stem-loop structure on RNA that causes RNA to slip off template. § 2. Rho (ρ) dependent termination: a protein called the ρ factor travels along the synthesized RNA and bumps off the polymerase Ribozymes, spliceosomes, small nuclear ribonucleoproteins (snRNPs), small nuclear RNAs (snRNAs) o Ribozyme = RNA molecule that is capable of catalyzing specific chemical reactions o snRNPs = RNA-protein complexes that combine with unmodified pre-mRNA and various other proteins to form a spliceosome, a large RNA-protein molecular complex upon which splicing of pre-mRNA occurs o snRNAs = complexed with proteins to form snRNPs to splice primary RNA transcripts. Functional and evolutionary importance of introns  o Introns and alternative splicing allow different mRNA sequences and ultimately a greater variety of proteins through translation. Without introns or alternative splicing, less protein variability. o Efficient way to generate wide variety of proteins through use of mRNA compared to modifying preexisting proteins. o Splicing of introns = using snRNA/snRNP to form a spliceosome complex to excise intron lariat loop Post-transcriptional modification o Post-transcriptional modifications include addition of the 5’ cap, polyA tail and splicing o In prokaryotes, mRNAs with better Shine-Dalgarno sequence are translated more o In eukaryotes, post-transcription regulation can involve adding more polyAs to mRNA (longer mRNA life time), modulation of the translation machinery (phosphorylation of initiation factors), or storing mRNAs to be translated at a later time (mRNA masking) o The cap and polyA tail are added co-transcriptionally, but still considered post transcriptional o Splicing gives rise to isoforms. Depending on how you arrange the introns/exons, you get different proteins by alternative splicing. Translation (BIO) Role of mRNA, tRNA, rRNA o mRNA: contains codons that code for the peptide sequence o tRNA: contains the anticodon on the “tip” and the corresponding amino acid on the “tail.” Link correct amino acid to its corresponding mRNA codon through codon-anticodon interaction. o rRNA: forms the ribosome. Catalyzes the formation of the peptide bond. Role and structure of ribosomes o Ribosome is the enzyme that catalyzes protein synthesis o Ribosome has 2 subunits - the large and the small o Large subunit responsible for peptidyl transfer reaction o Small subunit responsible for recognizing mRNA and binds to Shine-Dalgarno sequence on mRNA (Kozak sequence for eukaryotes) o Both subunits needed for translation to occur and they come together in a hamburger fashion that sandwiches the mRNA and tRNAs in between Mechanism of translation o 1. Chain Initiation: To begin, you need to form initiation complex, basically an assembly of everything. This includes mRNA, initiator tRNA (fmet), and ribosome (initiation factors, and GTP aids in formation of initiation complex). The initiation complex forms around initiation codon (AUG), which is just downstream of the Shine-Dalgarno sequence. The Shine-Dalgarno sequence is the “promoter” equivalent of translation for prokaryotes (Kozak sequence for eukaryotes). o 2. Chain Elongation: protein made from N terminus to C terminus. mRNA codons read from 5’ to 3’ end. Elongation consists of: § 1. Binding: new tRNA with its amino acid (tRNA + amino acid is called aminoacyl- tRNA) enters the A site. GTP and elongation factor required. § 2. Peptidyl transfer: attachment of the new amino acid to existing chain in P site. The already existing chain in P site migrates and attaches to the aminoacyl-tRNA in the A site. § 3. Translocation: the lone tRNA in the P site gets kicked off (E site), and the tRNA in the A site, along with peptide attached to it, moves into the P site. The mRNA gets dragged along also - the codon that was in the A site is now in P site after translocation. A site is now empty and ready for the binding of a new aminoacyl-tRNA to a new codon. Elongation factor and GTP required. o 3. Chain Termination: When a stop codon is encountered, proteins called release factors, bound to GTP, come in and block the A site. Peptide chain gets cleaved from tRNA in the P site. Peptide chain falls off and the whole translation complex falls apart.  o Amino acid activation: enzymes called aminoacyl-tRNA synthetases attach the correct amino acids to their corresponding tRNAs. ATP required. Post-translational modifications to protein o Modifications of the protein through addition of groups to the protein through covalent bonds or cleavage of the protein o Deals with activation/inactivation or enhancing the protein’s function o Examples include phosphorylation, glycosylation, and ubiquitination (inactivation by tagging protein for proteasome degradation) Eukaryotic Chromosome Organization (BIO) Chromosomal proteins o Histones: responsible for compact packing and winding of chromosomal DNA. DNA winds itself around histone octamers. o Non-histone chromosomal proteins: all the other proteins are lumped together in this group. Responsible for roles such as regulatory and enzymatic. Single copy vs. repetitive DNA o Single copy = DNA sequence that does not repeat (ex. ATCCGTAG) o Single copy holds most of the organism’s important genetic information. It is transcribed and translated and has a low mutation rate. o Repetitive DNA = DNA sequence that does repeat (ex. ATCCATCC) o Repetitive DNA found near the centromeres. They may contain genes that are transcribed/translated; however, there may be parts that are not transcribed/translated. They have higher mutation rate. o Highly repetitive DNA contains no genes so not transcribed/translated and very high mutation rate (ex. telomeres) Supercoiling o Supercoiling is a wrapping of DNA on itself as its helical structure is pushed even further toward the telomeres during replication. To alleviate the torsional stress and reduce risk of strand breakage, DNA gyrase (DNA topoisomerase II) introduces negative supercoils. Heterochromatin vs. euchromatin o Euchromatin is structured as loose beads on a string. The beads represent nucleosomes. The majority of DNA is in euchromatin form, as it’s generally under active transcription all the time. Note: Prokaryotes only have euchromatin as heterochromatin has a more complex structure. o Heterochromatin is densely packed, so like coiled beads on a string. It was thought that the genes here were inaccessible for transcription, but recent research says otherwise. Additionally, if some euchromatin is not being transcribed, it may be converted into heterochromatin, and vice-versa. Telomeres, centromeres o Telomere: the 2 ends of the chromosome o Centromere: a region on the chromosome, can be at the center or close to one of the ends. After replication, sister chromatids are attached at the centromere. During mitosis, spindle fibers are attached at the centromere and pulls the sister chromatids apart. Control of Gene Expression in Prokaryotes (BIO) Operon Concept, Jacob-Monod Model o Operon = a cluster of genes transcribed as a single mRNA. The numerous genes share a single common promoter region on the DNA sequence and are transcribed as a group. Two types of operons: inducible and repressible systems. o Jacob-Monod Model: § 1. Structural gene: codes for protein of interest § 2. Operator site: nontranscribable region of DNA capable of binding a repressor protein § 3. Promoter site: provides a place for RNA polymerase to bind  § 4. Regulator gene: codes for the repressor protein Gene repression in bacteria o Operons have a binding site for regulatory proteins that turn expression of the operon “up” or “down” o Some regulatory proteins are repressors that bind to pieces of DNA called operators. When bound to its operator, a repressor reduces transcription (e.g., by blocking RNA polymerase from moving forward on DNA) Positive control in bacteria o Some regulatory proteins are activators. When an activator is bound to its DNA binding site, it increases transcription of the operon (e.g., by helping RNA polymerase bind to the promoter) Control of Gene Expression in Eukaryotes (BIO) Transcriptional regulation o Transcription factors (protein) bind to enhancers or silencers (DNA) to affect transcription. Enhancers increase transcription when bound, while silencers decrease it. The main difference in eukaryotes from prokaryotes is that enhancers/silencers can be very far away from actual promoter and can be upstream or downstream. The DNA must loop back on itself so that the transcription factor bound to enhancer/silencer can make contact with promoter. Intermediate proteins are involved in the process. o Eukaryotes lack bacterial transcription regulation mechanisms such as the operon and attenuation DNA binding proteins, transcription factors o DNA-binding proteins and transcription factors bind to DNA. TFs have a DNA-binding domain. o DNA-binding domains interact with the grooves in the double helix (major and minor grooves) Gene amplification and duplication o Once the transcription complex is formed, basal (or low-level) transcription can begin and maintain moderate, but adequate, levels of the protein encoded by this gene in the cell. There are times, however, when the expression must be increased, or amplified, in response to specific signals such as hormones, growth factors, and other intracellular conditions. Eukaryotic cells accomplish this through enhancers and gene duplication. o Gene duplication can also increase expression of a gene product by duplicating the relevant gene. Genes can be duplicated in series on the same chromosome, yielding many copies in a row of the same genetic information. Genes can also be duplicated in parallel by opening the gene with helicases and permitting DNA replication only of that gene; cells can continue replicating the gene until hundreds of copies of the gene exist in parallel on the same chromosome. Post-transcriptional control, basic concept of splicing (introns, exons) o tRNA and rRNA modifications: some normal nucleotides are modified to control the structure of these RNAs o mRNA modifications § RNA splicing: sequences called introns cut out, sequences called exons are kept and spliced (joined) together § Alternate splicing: different ways of cutting up the RNA and rejoining the exons pieces makes different final RNA products § 5’ capping and 3’ poly-A tail: these help to protect the RNA from degradation, so they can last longer o After the correct modifications, RNA is transported out of nucleus where they can function in translation o After some time, RNA is degraded. The rate and timing of RNA degradation can be controlled by the cell. Cancer as a failure of normal cellular controls, oncogenes, tumor suppressor genes o Failure of normal cellular controls: § Cancer cells continue to grow and divide in situations normal cells would not § Cancer cells fail to respond to cellular controls and signals that would halt this growth in normal cells  § Cancer cells avoid apoptosis (self-destruction) that normal cells undergo when extensive DNA damage is present § Cancer cells stimulate angiogenesis (cause new blood vessels to grow to nourish the cancer cell) § Cancer cells are immortal while normal cells die after a number of divisions § Cancer cells can metastasize - break off and grow in another location o Oncogenes: genes that cause cancer when activated. The product of many oncogenes is involved in speeding up cell division. Before an oncogene is activated, it is a harmless proto-oncogene. Something occurs that changes proto-oncogene to an oncogene. Classic example is src. o Tumor suppressors: if the oncogene is the “bad” gene, tumor suppressors are the “good” genes. The product of many tumor suppressors is involved in slowing down or controlling cell division. If something happens that cause tumor suppressor to no longer function, then the cell becomes cancerous. Classic example is p53. Regulation of chromatin structure o Chromatin made up of DNA, histone proteins, and non-histone proteins. There are repeating units of chromatin, called nucleosomes, which are made up of double helical DNA wrapped around a core of eight histones. o DNA comes in two flavors: densely packed, and transcriptionally inactive DNA called heterochromatin, and the less dense, transcriptionally active DNA called euchromatin o Methylated DNA may be bound to methyl cpg-binding domain proteins that recruit additional proteins to the locus certain genes and other chromatin remodeling proteins, and this modifies the histones, forming condensed inactive heterochromatin that is basically transcriptionally silent. o Acetylation promotes open DNA (aka active chromatin or euchromatin) DNA methylation o Involved in chromatin remodeling and regulation of gene expression levels in the cell. Methylation often linked with silencing of gene expression. During development, methylation plays an important role in silencing genes that no longer need to be activated. o Heterochromatin regions of the DNA are more heavily methylated, hindering access of the transcriptional machinery to the DNA. Role of non-coding RNAs o Non-coding RNA is a functional RNA transcribed from DNA but not translated into proteins. They function to regulate gene expression at the transcriptional and post-transcriptional level. o Three major classes are microRNAs (miRNAs), short interfering RNAs (siRNAs), and piwi- interacting RNAs (piRNAs) Recombinant DNA and Biotechnology (BIO) Gene cloning o Retrieve gene of interest and plasmid that has one area with similar sequence. Cut both with the same Restriction Enzyme. Hybridize, then seal with DNA ligase. This produces a Recombinant Plasmid. Insert plasmid into bacteria and allow for replication inside bacteria. o Plasmid must have a restriction site because you need to open it up for the insertion of your gene. o Plasmid must have an origin of replication because you want to clone your gene, which is inside your plasmid. o Plasmid must have antibiotic resistant gene because this lets you kill competing, useless bacteria that don’t have your plasmid. when you add an antibiotic, only the bacteria with antibiotic resistant plasmid will live. o Plasmids replicate independently of the genomic DNA of the bacteria. Restriction enzymes o Restriction enzymes cut double stranded DNA at palindrome sequences. The resulting fragments are called restriction fragments. o If you read from 5’ à 3’ of one strand, then read from 5’ à 3’ of the other strand, and they are the same, then the section of the double stranded DNA that you read is palindrome sequence.  o Some restriction enzymes cut to make sticky ends, which can hybridize. o Some restriction enzymes cut to make blunt ends, which cannot hybridize. DNA libraries o DNA library is a collection of DNA fragments that have been cloned into vectors so that researchers can identify and isolate the DNA fragments that interest them for further study. There are two kinds of libraries: genomic and cDNA libraries. o Genomic libraries contain large fragments of DNA in either bacteriophages or bacterial or P1- derived artificial chromosomes (BACs and PACs). o cDNA libraries are made with cloned, reverse-transcribed mRNA, and therefore lack DNA sequences corresponding to genomic regions that are not expressed, such as introns and 5’ and 3’ noncoding regions. cDNA libraries generally contain much smaller fragments than genomic DNA libraries and are usually cloned into plasmid vectors. Generation of cDNA o Once mRNA is purified, oligo-DT (a short sequence of deoxy-thymidine nucleotides) is tagged as a complementary primer which binds to the poly-A tail providing a free 3’-OH end that can be extended by reverse transcriptase to create the complementary DNA strand. Now, the mRNA is removed by using RNAse enzyme leaving a single stranded cDNA (sscDNA). This sscDNA is converted into double stranded DNA with the help of DNA polymerase. o However, for DNA polymerase to synthesize a complementary strand a free 3’-OH end is needed. This is provided by sscDNA itself by generating a hairpin loop at the 3’ end by coiling on itself. The polymerase extends the 3’-OH end and later the loop at 3’ end is opened by the scissoring action of S1 nuclease. Restriction endonucleases and DNA ligase are then used to clone the sequences into bacterial plasmids. o The cloned bacteria are then selected, commonly through the use of antibiotic selection. Once selected, stocks of the bacteria are created which can later be grown and sequenced to compile the cDNA library. Hybridization o Hybridization, also called annealing, is where DNA strands base pair with each other. o In Southern blotting, DNA probes are used to hybridize onto DNA fragments containing a target sequence. o In gene cloning, hybridization refers to the process where sticky ends from a restriction fragment of a gene base pairs with the same sticky ends on a plasmid. Expressing cloned genes o cDNA transformed into plasmid, then add antibiotic resistant gene. o Infect bacteria with plasmid and add antibiotics. This allowed only the successfully infected bacteria to survive which contain our gene of interest. o The bacteria replicates creating many copies of our gene of interest. Polymerase chain reaction o 1. Denaturation: heat (90˚C) to separate double stranded DNA template o 2. Annealing: cool reaction in order for primers to anneal to the now single stranded DNA template § Excess amount of primers, so they outcompete re-annealing of the template strands o 3. Elongation: use heat stable polymerase to extend the primers o 4. Repeat steps 1 to 3 for n cycles. The resulting amplification of the original DNA template after n cycles is 2n Gel electrophoresis and Southern blotting o Gel electrophoresis: separation of proteins, DNA, or RNA based on size and/or charge. For proteins and small molecules of DNA and RNA, the gel will be polyacrylamide. For larger molecules of DNA (> 500bp), the gel will be agarose. An electrical charge is placed across the gel. At the bottom is the positively charged anode and the top is the negatively charged cathode. § Keep in mind, since a voltage source is applied to gel electrophoresis, it follows the same principles as an electrolytic cell. Negatively charged molecules will travel toward the  anode. Because of resistance of the gel, larger molecules will have a harder time moving and thus, the molecules will be separated by size with the smallest molecules toward the bottom. The gel can be stained for visualization, typically using Coomassie Blue dye. A lane will be loaded with a collection of molecules of a known size, called a ladder, which can be used to determine the size of the molecules being ran. o Southern blotting: § 1. DNA strand of interest is exposed to restriction enzymes that cut the DNA strand into smaller fragments. § 2. The newly cleaved strands of DNA are denatured using a solution of NaOH to create ssDNA strands § 3. The single stranded cleaved strands of DNA undergo gel electrophoresis, separating them by size. Smaller fragments will be found at the bottom of the gel. Polyacrylamide is used if the stands are less than 500 base pairs. Agarose is used if the strands are over 500 base pairs. § 4. The DNA from the gel is transferred to a sheet of nitrocellulose paper and then exposed to a 32P radiolabeled DNA probe that is complementary to DNA of interest. § 5. Nitrocellulose paper is then viewed using autoradiography to identify the strand of interest. DNA (Sanger) sequencing o Used to determine the sequence of nucleotides in a strand of DNA o Modified nucleotides, known as “dideoxynucleotides” (ddNTPs), are used in this method. ddNTPs are missing the OH group on the 3’ carbon, thus unable to create a new 5’à3’ phosphodiester bond. This allows us to control termination of replication. o 1. DNA strand of interest is denatured using an NaOH solution to create a ssDNA strand that we can use for replication o 2. ssDNA strand of interest is added to a solution containing: § 1. A radiolabeled DNA primer that is complementary to the gene of interest § 2. DNA polymerase § 3. All four dNTPs (dATP, dTTP, dCTP, dGTP) § 4. A very small quantity of a single ddNTP (e.g., ddATP) § This is done once for each of the four nucleotides in separate solutions o 3. Each solution containing a specific dNTP and ddNTP are placed in their own lane of a gel and ran under gel electrophoresis. The gel is transferred to a polymer sheet and autoradiography is used to identify the strands in the gel. o For each respective nucleotide, the insertion of a ddNTP will terminate replication and create various strands of different length that correspond to that specific nucleotide. Therefore, the gel can be read from bottom-to-top to determine the nucleotide sequence. The smaller the fragment, the further it travels in the gel. Analyzing gene expression o Northern blotting: Similar steps to Southern except Northern uses RNA so steps 1 and 2 are not done. o Western blotting: Detection of a specific protein in a sample. § 1. Proteins from a sample are loaded into an SDS-PAGE gel and separated based on size. § 2. Proteins from gel are transferred to a polymer sheet and exposed to a radiolabeled antibody (sometimes using two antibodies; one specific to protein of interest and another radiolabeled antibody that binds to first antibody) that is specific to protein of interest § 3. The polymer sheet is viewed using autoradiography. The protein of interest that is bound to the radiolabeled antibody will be visible. o RT-qPCR: used when starting material is RNA. In this method, RNA is first transcribed into complementary DNA (cDNA) by reverse transcriptase from total RNA or messenger RNA (mRNA). cDNA then used as template for qPCR reaction. RT-qPCR is used in a variety of applications including gene expression analysis, RNAi validation, microarray validation, pathogen detection, genetic testing, and disease research.  § RT-qPCR can be performed in a one-step or a two-step assay. One-step assays combine reverse transcription and PCR in a single tube and buffer, using a reverse transcriptase along with a DNA polymerase. One-step RT-qPCR only utilizes sequence-specific primers. In two-step assays, the reverse transcription and PCR steps are performed in separate tubes, with different optimized buffers, reaction conditions, and priming strategies. Determining gene function o Knocking out the gene allows us to observe what functions the gene are responsible for. Stem cells o Totipotent: can differentiate into any cell of an organism, including the placenta, the amnion and chorion o Pluripotent: can give rise to all cell types, excluding the placenta, the amnion and chorion. They arise from Totipotent cells, and are more specialized o Multipotent: can develop into more than one cell type, but are more limited than pluripotent cells; adult stem cells and cord blood stem cells are considered multipotent o Embryonic: within first couple of cell divisions after fertilization are the only cells that are totipotent Practical applications of DNA technology: medical applications, human gene therapy, pharmaceuticals, forensic evidence, environmental cleanup, agriculture o Medical applications: some vaccines we use recombinant DNA technology including hep B virus and the herpes virus and malaria. o Gene therapy: intended for diseases in which a given gene is mutated or inactive, giving rise to pathology. By transferring a normal copy of the gene into the affected tissues, the pathology should be fixed. § Efficient gene delivery vectors must be used to transfer the cloned gene into the target cells’ DNA. Because viruses naturally infect cells to insert their own genetic material, most gene delivery vectors in use are modified viruses. A portion of the viral genome is replaced with cloned gene such that the virus can infect but not complete its replication cycle. o Forensics: There are parts of the genome known as non-coding regions of the genome. These regions can help forensic scientists identify specific individuals by looking at things like short tandem repeats, STRs, which are short sequences of DNA, two to six base pairs long. They are found in high amounts and to varying degrees between individuals. Thus, if they sequence these STRs, they could identify specific individuals given a DNA sample. o Agriculture: scientists can now create crops resistant to insects and resistant to herbicides. Some can delay ripening of crops, so you can transport crop from farm to store. Safety and ethics of DNA technology o Safety concerns such as increased resistance in viruses and bacteria can impact both humans the environment in which we live. Ethical dilemmas arise such as testing for life-threatening genetic diseases and potentially terminate a pregnancy based on results. How much should we meddle with our own genetic makeup becomes an issue. Content Category 1C: Transmission of heritable information from generation to generation and the processes that increase genetic diversity Mendelian Concepts (BIO) Phenotype and genotype o Phenotype = what you observe (i.e. height, color, whether organism exhibits trait) o Genotype = the genetic makeup (i.e. homozygous dominant, heterozygous, homozygous recessive) Gene o Stretch of DNA that codes for a trait (in molecular bio, a gene codes for a protein, which acts to bring about a trait) Locus o Location of a gene on a chromosome Allele: single and multiple o Allele is a variant of a gene (gene may have a number of alleles, all alleles of the same gene exist at the same locus) o Cell holds two alleles for each gene, one from mom and one from dad o 2 alleles = simple case (i.e. TT, Tt, tt) o When gene has more than 2 alleles, it is called MULTIPLE ALLELES § e.g. Blood type (have IA, IB, i) Homozygosity and heterozygosity o Homozygous = when two alleles that an individual carry are the same (i.e. AA, aa) o Heterozygous = when two alleles that an individual carry are different (i.e. Aa) Wild-type o The “normal” allele or phenotype for an organism (usually most prevalent, but not always) Recessiveness o The “weak” allele o Only expressed if both copies are present (aa) Complete Dominance o Normal way of assigning alleles § I.e. AA = dominant; Aa = dominant; aa = recessive Co-dominance o When the heterozygous conveys both traits § I.e. Blood type: AA = expresses A; AB = expresses A and B; BB = expresses B Incomplete dominance, leakage, penetrance, expressivity o Incomplete Dominance: § When the heterozygous conveys a mixture of the two alleles I.e. AA = expresses A; AB = expresses a MIX of A and B; BB = expresses B I.e. When crossing black and white chickens, you make blueish gray chickens o Leakage § Gene flow from one species to another (through hybrid offspring) o Penetrance § Frequency that a genotype will result in a phenotype § e.g., 100% penetrance means that if you have the genes for being smart, you will 100% be smart Less than 100% means that you could have the genes for being smart, but that doesn’t mean 100% that you’ll be smart o Expressivity § The degree to which a penetrant gene is expressed § Constant Expressivity = if gene for being smart manages to penetrate (show up as a trait), then IQ is 120  § Variable Expressivity = your IQ does not have to be 120, it could be somewhat lower or higher Hybridization: Viability o Process of two complementary, single-stranded DNA or RNA combining together, producing a double-stranded molecule through base pairing o Technique is used for interbreeding between individuals of genetically distinct populations Gene Pool o All the alleles in a population o The sum of all genes/alleles in a population at a given time o High ratings have more genetic diversity and more fitness Meiosis and Other Factors Affecting Genetic Variability (BIO) Significance of meiosis o Genetic division process of creating haploid sex cells o Occurs in two separate mitosis events o Final result = 4 cells with n number of singular chromosomes o Introduces genetic variability by genetic recombination § Genetic recombination = product of independent assortment and crossing over, which introduces genetic variability Important differences between meiosis and mitosis Mitosis Meiosis No tetrad Tetrad formation (pairing of homologous chromosomes) and crossing over Daughter cells identical to parent cells Daughter cells different from parent cells Diploid (2n) daughter cells Haploid (n) daughter cells 1 division involved 2 divisions involved 2 daughter cells 4 sperm cells or 1 egg (with polar bodies) *A human cell has 46 total or 23 pairs of chromosomes. Following mitosis, the daughter cells would each have a total of 46 chromosomes. After meiosis I, the two daughter cells would have 23 chromosomes Segregation of genes o Independent Assortment  § Generates genetic variation § Cell has 2 copies of each somatic chromosome – one from mom/dad Independent assortment shuffles the chromosomes, and then places only one copy of each into the gamete e.g., AaBb - A and B alleles assort independent of each other § Mechanism: During metaphase I of meiosis, homologous chromosome pair up along the metaphase line in random orientation – sometimes the mom chromosome is on the right, or sometimes on the left During anaphase I of meiosis, the homologous chromosomes are pulled apart o Those on the left will go into one daughter cell, the one on the right will go into another daughter cell o Linkage § Because of independent assortment, genes on different chromosomes can be randomized (HOWEVER, genes on same chromosome cannot be randomized by this mechanism) § Genes on same chromosome are linked to a certain extent Basically, genes located near one-another on the same chromosome are likely to be inherited together § Crossing Over = mechanism that reduces linkage Only efficient when the genes are physically apart from each other on the chromosome § When genes are further apart on a chromosome, crossing over makes them less linked § The physically closer the genes are, the more linked they are o Recombination (made up by independent assortment and crossing over, increase genetic diversity) § Single Crossovers Occurs during prophase I and suggests that chromatids exchange alleles at a locus (results in genetic recombination) When homologous chromosomes are aligned and chromatids from two different chromosomes can exchange segments resulting in genetic recombination Only affect the ends of a chromosome’s arms § Double Crossovers An event that has 3 outcomes: o Chromatids exchange alleles, then exchange back (no R) o Chromatids exchange alleles, then exchange with different chromatids (R) o Chromatids exchange alleles, then two different chromatids on the same chromosome exchange again Chromatids from two homologous chromosomes come in contact at two points  Can affect segments of the chromosome closer to the middle of the chromosome § Synaptonemal Complex Complex of proteins that are located between pairs of homologous chromosomes Protein structure that mediates synapsis (the pairing of homologous chromosomes in Prophase I) o Consists of SYCP2 and SYCP3 which attach laterally to homologous chromatin structures and are attached via a central region (SYCP1 and other proteins) – interdependent with recombination § Tetrad A chiasma (point at which paired chromosomes remain in contact during the first metaphase of meiosis, and at which crossing over and exchange of genetic material occur between the strands) between a pair of homologous chromosomes resulting in the formation of 4 chromatids o Sex-linked characteristics § Gene for characteristic is on the X chromosome § Mom always donates X chromosome o Very few genes on Y chromosome § Y chromosome is very small and carries few genes of importance § All the sex-linked alleles are carried on the X chromosome o Sex determination § XX = female § XY = male o Cytoplasmic/extra-nuclear inheritance § Cytoplasmic inheritance = inheritance of things other than genomic DNA § All cellular organelles, such as mitochondria, is inherited from the mother § Extra-nuclear inheritance = situations where genes are inherited outside of the nucleus Includes receiving all the information about the mitochondria from the mother’s egg Mutation o General concept of mutation – error in DNA sequence § Mutation = change in DNA by means other than recombination o Types of mutations: random, translation error, transcription error, base substitution, inversion, addition, deletion, translocation, mispairing § Random:  Random changes in the DNA sequence (could be due to radiation, chemicals, replication error, etc.) § Translation Error: Even if the DNA for a gene is perfect, errors during translation can cause expression of a mutant phenotype § Transcription Error: Even if the DNA for a gene is perfect, errors during transcription can cause expression of a mutant phenotype § Base Substitution: Mutation involving a base (A, T, G, C) changing to a different base § Inversion: A stretch of DNA (segment of chromosome) breaks off, then reattaches in the opposite orientation o Chromosome rearrangement in which a segment of the chromosome is reversed end to end (i.e. when a chromosome breaks and rearranges within itself) § Addition: Also called insertion à an extra base is added/inserted into the DNA sequence (can cause frameshift mutation) § Deletion: A base is taken out of the DNA sequence (can cause frameshift mutation) § Translocation: A stretch of DNA (segment of chromosome) breaks off, and then reattaches somewhere else § Mispairing: A not pairing with T, or G not pairing with C o Advantageous vs. deleterious mutation § Advantageous = results in a benefit in the fitness of an organism § Deleterious = results in harmful effects on the fitness of an organism o Inborn errors of metabolism § Genetic diseases resulting in faulty metabolism § Considered congenital (present since birth) metabolic diseases & inherited metabolic diseases o Relationship of mutagens to carcinogens § Mutagen = something that causes a mutation § Carcinogen = something that causes a mutation that causes cancer § Carcinogens are almost always mutations (EXCEPTION: some are direct mitogens = increase mitosis) § Not all mutagens are carcinogens Genetic Drift o Random changes in a population (random changes in allele frequencies) o Effect of genetic drift increases as the population size decreases Synapsis or crossing-over mechanism for increasing genetic diversity o Synapsis and crossing over help increase genetic diversity (genes recombine and create larger diversity in number of recombinants) Analytic Methods (BIO) Hardy-Weinberg Principle o p+q=1 o (p + q)2 = 1 à p2 + 2pq + q2 = 1 o 5 Assumptions of Hardy-Weinberg: § Infinitely large population  § No mutation § No migration § Random mating (no sexual selection) § No natural selection Testcross (Backcross; concepts of parental, F1, and F2 generations) o Test Cross § So, you have something with dominant phenotype. It could either be Aa or AA. To find out, you cross it with the homozygous recessive aa. If Aa, half of the offspring will express the recessive phenotype. If AA, no offspring will express the recessive phenotype o Back Cross § Matting between the offspring and the parent = preserve parental genotype o Parental Generation = P § Generation of the parent. On a pedigree, this is the row that represents the parents o F1 Generation = Filial 1 = children § On a pedigree, this is the row below the parents, and represents the children of the parents o F2 Generation = Filial 2 = grandchildren § On a pedigree, this is the row below the F1, and represents the children of the F1 and the grandchildren of the parents Gene mapping: crossover frequencies o Genetic recombination occurs between maternal and paternal sister chromatids § Can also occur with the identical chromatid within one chromosome, but that will not have any consequence o More likely for recombination to occur over a larger distance (can exchange genetic information anywhere along the long stretch of chromatid) § Further apart that two genes are, the more likely it is that they recombine o Less likely for recombination to occur over a smaller distance (not that much space) o Centimorgen = unit of measurement we use to measure distance on a chromosome (or genetic map unit) § Distance between genes for which one product of meiosis in 100 is recombinant § e.g., 1 distance is 25 m.u. ; another is 6 m.u. For 25 m.u., 25% of the time that meiosis happens, recombination will occur with respect for the two genes within the map unit distance For 6 m.u., 6% of the time that meiosis happens, recombination will occur Biometry: statistical methods o Use of statistical methods to understand biological data o ID genes in the population that are bad Evolution (BIO) Natural selection o Fitness concept § Fitness is defined as the ability to pass your genes on (reproductive success) o Selection by differential reproduction § Individuals who produce more VIABLE offspring are selected for § Individuals who reproduce LESS VIABLE offspring are selected against o Concepts of natural and group selection § Natural Selection: Survival and reproduction of the fittest § Directional Selection: selects for a trait of one extreme (e.g., selection for tall canopy tree in rainforest because the taller tree can reach the sun) § Stabilizing Selection: selects for a trait that is moderate, and selects against the extremes (e.g., just the right birth weight when a baby is born) § Disruptive Selection: selects for the extremes (e.g., birds occupying habitat with 2 different niches, one bird with small beak, the other with big beak)  § Group Selection: natural selection acting on the group, not the individual (altruism = sacrifice the fitness of the individual to benefit the group or family, which shares similar genes with the individual) When benefit outweighs the cost, altruistic behavior is selected for o Evolutionary success as increase in percent representation in the gene pool of the next generation § If the frequency of an allele is increased, then that’s evolutionary success for that allele § If the frequency of alleles increased in a population, then that’s evolutionary success for that individual Speciation o Polymorphism § Different forms for alleles or traits (i.e. a yellow jaguar and a black jaguar) o Adaptation and specialization § Adaptation = genetic change in a population caused by natural selection § Specialization = adaption of traits to better fill a niche o Inbreeding § Mating between relatives § Increases the frequency of homozygotes, decreases heterozygotes, and decreases genetic diversity § Inbreeding depression occurs because of the increase in frequency of homozygous recessive detrimental alleles o Outbreeding § Mating with non-relatives, which is just the opposite of inbreeding § Increases heterozygosity o Bottleneck § Severe reduction in population size (e.g., can be caused by natural disaster that wipes out majority of the population) § Genetic drift = random changes in allele frequencies § Effect of genetic drift increases as population size decreases § Bottlenecks increase the effect of genetic drift Evolutionary Time o Random genetic mutations (drift) that are not acted on by natural selection (neutral) occur at a constant rate o By measuring the amount of these neutral mutations, you can find out how much time has passed o You can compare genome differences between two species to find out how long ago they diverged o “Molecular Clock” Content Category 1D: Principles of bioenergetics and fuel molecule metabolism Principles of Bioenergetics (BC, GC) Bioenergetics/thermodynamics o Free energy/Keq: Free energy is the energy available that can be converted to do work ∆G = ∆H - T∆S T is temperature in Kelvin § Equilibrium constant (2 ways of getting Keq) From an equation, Keq = products/reactants = [C]c[D]d/[A]a[B]b From thermodynamics, ∆G˚ = -RT ln (Keq) o Derivation: ∆G = 0 at equilibrium o ∆G = ∆G˚ + RT ln Q o 0 = ∆G˚ + RT ln Qat equilibrium o ∆G˚ = -RT ln Qat equilibrium At equilibrium: o ∆G = 0 o rforward = rbackward o Q = Keq Keq is ratio of kforward over kbackward o If Keq > 1, then position of equilibrium is to the right, more products present at equilibrium o If Keq = 1, then position of equilibrium in center, number of products roughly equal to number of reactants o If Keq < 1, then position of equilibrium is to the left, more reactants are present at equilibrium § Relationship of the equilibrium constant and ∆G˚ ∆G = ∆G˚ + RT ln Q o Set ∆G = 0 at equilibrium o Q becomes Keq at equilibrium 0 = ∆G˚ + RT ln (Keq) ∆G˚ = -RT ln (Keq) o Concentration § Le Châtlier’s Principle: if you knock a system off its equilibrium, it will readjust itself to reachieve equilibrium § A reaction at equilibrium doesn’t move forward or backward, but the application of Le Châtlier’s principle means that it will proceed forward or backward in order to restore equilibrium o Endothermic/exothermic reactions § Endothermic = energy taken up by reaction in form of heat. ∆H is positive § Exothermic = energy released by reaction in form of heat. ∆H is negative § Enthalpy, H, and standard heats of reaction and formation Enthalpy, H, is heat content of a reaction.  ∆H is change in heat content of a reaction. + means heat is taken up, - means heat is released Standard heat of reaction, ∆Hrxn, is change in heat content for any reaction Standard heat of formation, ∆Hf, is change in heat content of a formation reaction Formation reaction is where a compound or molecule in its standard state is formed from its elemental components in their standard states. Standard state is where things are in their natural, lowest energy, state. e.g. oxygen is O2 (diatomic gas) and carbon is C (solid graphite) Unit for enthalpy is energy (J), or can be expressed as energy per mol (J/mol) o Free Energy: G § Free energy is energy available that can be converted to do work § ∆G = ∆H - T∆S o Spontaneous reactions and ∆G˚ § Spontaneous reactions occur by themselves. § They have negative ∆G. § Do not assume that an exothermic reaction is spontaneous, because a large, negative ∆S can cause it to become nonspontaneous § Do not assume that an endothermic reaction is nonspontaneous, because a large, positive ∆S can make it spontaneous § Do not assume that spontaneous reactions will occur quickly, because it depends on kinetics Phosphoryl group transfers and ATP o ATP hydrolysis ∆G 7 If the acid is stronger than the base, then equivalence is COOH > R, then L=S and D=R. For example, L-Alanine = S- Alanine § If the priority of NH2 >R > COOH, then L=R, and D=S. For example, L-Cysteine = R- Cysteine § L-amino acids are more common in nature, and are the type found in proteins. D-amino acids are less common in nature, and are never found in proteins o Dipolar ions § At low pH, amino acids exist in the cationic form § At high pH, amino acids exist in the anionic form § At pH = pI, amino acids exist in the zwitterion form, which is overall neutral o Classification § Acidic or basic If the R group contains carboxylic acid, then it’s an acidic amino acid. There are two acidic aa: aspartic and glutamic acid If the R group contains an mine group, then it’s a basic amino acid. There are three basic aa: lysine, arginine, and histidine § Hydrophilic or hydrophobic Hydrophobic: If the R group doesn’t contain any of the stuff below Hydrophilic: IF the R group contains acids, bases, amines or alcohols  o Synthesis of α-amino acids (OC) § Strecker Synthesis Starting material: R-aldehyde Reagents: cyanide (KCN), ammonium (NH4Cl) § Gabriel Synthesis Starting material: R-halide Reagents: 1. phthalimide, 2. NH2-NH2 Product: Amino acid with the -R group originally on the halide Peptides and proteins: reactions o Sulfur linkage for cysteine and cysteine § Cysteine = side chain with the thiol group § Cystine = 2 cysteines forming a disulfide bond o Peptide linkage: polypeptides and proteins § Peptide bond = amide bond § Peptide bond is formed by amine group attacking the carbonyl carbon o Hydrolysis (BC) § Peptide bond is very difficult to hydrolyze. It requires a strong base, or a biological enzyme General Principles o Primary structure of proteins § Primary structure = sequence § Primary structure of proteins is read from N-terminus to C-terminus o Secondary structure of proteins § Secondary structure = repetitive motifs formed by backbone interactions § Backbone interactions = hydrogen bonding between the NH and C=O § Two most common secondary structures are α helices and β pleated sheets § The α helix is right-handed, with the R groups sticking outward § In β sheets, R groups stick out above and below the sheet o Tertiary structure of proteins § 3D structure of proteins § Caused electrostatic side chain - side chain interactions o Isoelectric point § pH at which the molecule is neutral § Acidic amino acids and proteins with lots of acidic side chains have a lower isoelectric point § Basic amino acids and proteins with lots of basic side chains have a higher isolectric point The Three-Dimensional Protein Structure (BC) Conformational stability o Hydrophobic interactions § Hydropathy plot Used to measure number of hydrophobic regions in a multidomain protein Region with positive hydropathy index indicates a hydrophobic region o Solvation layer (entropy) § Nonpolar aa’s pushed towards inside of a protein. Solvation layer of water forms around proteins Quaternary structure o Separate chains/subunits joining together o Caused by covalent disulfide bonding of cysteine side chains Denaturing and Folding o Amino acid sequence vital for folding  o Factors causing denaturing § Urea à disrupts H-bonding § Salt/pH change à disrupts ionic bonds § Mercaptoethnaol à disrupts H-bonding § Organic solvents à disrupts hydrophobic forces § Heat à disrupts all forces Non-Enzymatic Protein Function (BC) Two protein types: globular and structural o Globular: for enzymes, receptors, channels, transport o Structural: made of long polymers, used for structure, movement Binding o Special feature of some proteins is the capability to bind other molecules with non-covalent interactions o Protein binding characterized by affinity and specificity for the binding target Immune System o High degree of protein variability allows for a key feature of the adaptive (or acquired) immune system, the production of antibodies o An antibody is a type of protein that has a unique and very specific binding site that will readily bind its target, called an antigen, such that its target is inactivated or tagged for immune response o Glycoproteins: proteins with carbs attached. Includes antigens on red blood cells Motor o Motor protein can perform mechanical work by coupling exergonic (energy releasing) ATP hydrolysis to a conformational change that allows for interaction with the protein’s target substrate o Muscle contraction achieved through a process of the motor protein myosin binding and releasing its microfilament (actin) substrate o Myosin also acts on microfilaments of the cytoskeleton to generate cellular movement o Two other types of motor proteins, kinesins and dyneins, act on microtubules and play a role in transport within the cell o Kinesin is microtubule “tracks” to deliver cellular cargo (e.g. chromosomes during mitosis), generally in an anterograde direction (center to periphery) o Dynein is used in retrograde cargo transport in the axons of neurons, and is capable of sliding microtubules in relation to one another, generating the movement of cilia and flagella Lipids (BC, OC) Description, Types o Storage § Triacylglycerols Glycerol + 3 Fatty acids à Triacylglycerol The reverse of triacylglycerol synthesis is saponification § Free fatty acids: saponification Saponification is the ester hydrolysis of triacylglycerols using a strong base Traditionally, the base that is used is lye, the common name for sodium or potassium hydroxide Result is basic cleavage of the fatty acid, leaving the sodium salt of the fatty acid and glycerol The fatty acid salt is known as soap o Structural § Phospholipids and phosphatides Lipids with phosphate group attached Amphipathic, can form micelles  § Sphingolipids (BC) Phosphoglycerides but with a sphingosine backbone. In cell membrane § Waxes Ester linkage between long chain alcohol and fatty acids. Very water-repellant o Signals/cofactors § Fat-soluble vitamins Include vitamins A, D, E, and K Dissolve in fat and can be stored in your liver and fat tissue until needed Vitamin A, or carotene, important in vision, growth and development, and immune function o Most significant metabolite of vitamin A is the aldehyde form, retinal, which is a component of the light-sensing molecular system in the human eye o Carotene = carrots high in vitamin A, known to improve vision Vitamin D, or cholecalciferol, can be consumed or formed in a UV-driven reaction in the skin. o In liver and kidneys, vitamin D is converted to calcitriol, the biologically active form of vitamin D o Calcitriol increases calcium and phosphate uptake in the intestines, which promotes bone production o Lack of vitamin D can result in rickets, condition seen in children and characterized by underdeveloped, curved long bones as well as impeded growth o Vitamin D regulates calcium, remember it is frequently added to milk to aid in absorption of calcium Vitamin E o Characterized by substituted aromatic ring with long isoprenoid side chain and are characteristically hydrophobic o Aromatic ring reacts with free radicals, destroying them o This prevents oxidative damage, an important contributor to the development of cancer and aging Vitamin K o Vital to posttranslational modifications required to form prothrombin, and important clotting factor in the blood o Aromatic ring of vitamin K undergoes a cycle of oxidation and reduction during the formation of prothrombin o Also required to introduce calcium-binding sites on several calcium- dependent proteins o Vitamin K for Koagulation § Steroids Nonpolar and can travel across the plasma membrane Type of lipid = nonpolar molecule Metabolic derivatives of terpenes Characterized by having four cycloalkane rings fused together: three cyclohexane and one cyclopentane Steroid functionality determined by oxidation status of these rings, as well as functional groups they carry Steroid hormones, secreted by endocrine gland, travel on protein carriers to distant sites where they bind to specific high-affinity receptors and alter gene expression levels § Prostaglandins (BC)  Regulate the synthesis of cyclic adenosine monophosphate (cAMP), which is a ubiquitous intracellular messenger In turn, cAMP mediates the actions of many other hormones Downstream effects include powerful effects on smooth muscle function, influence over the sleep-wake cycle, and the elevation of body temperature associated with fever and pain Carbohydrates (OC) Description o No