question

Pathology

answer

study of disease
clinical manifistations(signs, sympt, lab findings)
structural changes
molecular chanages
how do molecular changes result in observed clinical manisfestations

question

Hutchinson-Gilford Progeria Syndrome(HGPS)
Phenotype

answer

baldness
swollen veins
absence of eyebrowns and eyelashes
beak shaped nose
shrunken chin
narrow chest and swollen and stiff joints
narrowing of coronary arteries(premature cardiac disease)

question

Hutchington-Gilford Progeria Syndrome (HGPS)

Nucleus

answer

abnormal lobulated nuclei
lack of usual heterochromatin clumping on edge
thick electron dense area adjacent to the nuclear membrane where lamins are present
abnormal amounts of lamin A (LMNA gene) are present

question

Lamin

answer

an intermediate filament that polymerizes along the inner nuclear membrane which has a structural and regulatory role

question

Inofarnib

answer

an farnesyltransferase inhibitor that blocks production of farnysl-progerin
developed as a cancer chemotherapy agent but has some effect in-vitro and in phase 1 and 2 of clinical trials
used to treat HGPS

question

Treatment of HGPS

answer

farnesyltransferase inhibitor (FTI) like inofarnib
using small, interfering RNA molecules to interfere with the transcription of the mutant allele
siRNA exon skipping might also be useful to eliminate the site to which the farnesyl moiety is bound

question

Amino Acid Structure

answer

central ?-carbon
carboxyl group (coo-)
amino group (NH3+)
hydrogen
Side chain (R)

question

Chirality of Amino Acids

answer

L-amino acids the hydrogen is on the right
D amino acid the Hydrogen is on the left
all chiral except glycine
most mamalian amino acids we are interested in are L amino acids

question

Peptide Bond Formation

answer

condensation reaction btw COO- and NH3+
usually R groups are in trans- location where one is up and the others down
if they can interact and structure allows it then they can be in cis?

question

Weak Acid Vs Strong Acid

answer

weak acid is incomplete dissociation in water
strong acid is complete dissociation in water

question

Henderson-Hasselbalch Equation

answer

pH=pKa+log₁₀ [A-]/[HA]

question

pK_a of ?COOH and ?NH3

answer

?COOH = 1.8
?NH3 = 9.3

question

Amino Acid R-Group
Interactions

answer

hydrophobic interaction (ex phenylalanine side chains)
hydrogen bonds (H-O usually)
electrostatic interactions or ionic bonds(salt bridges)

question

Primary Structure

answer

order of amino acid residues in a polypeptide chain or protein
determined by the sequence of codons in mRNA resulting from genomic transcription and splicing

question

Start Codon

answer

AUG
methionine
usually cleaved off by enzymes after translation

question

Non-critical variation of structure

answer

these are called hidden variations
this is often the case with conservative substitution(amino acids that are similar
same function and same structure

question

Critical to structure variation

answer

different structure and different function
increases activity or decreases activity
or alters structural integrity (may be subject to cellular proteolysis)

question

Polymorphisms

answer

sequence variations in an allele within a population
sometimes important sometimes not

question

Developmental Variation

answer

certain variants of a protein family are differentially expressed based on the developmental stage of the organism
the variant proteins are called isoforms or isozymes
example are different forms of hemoglobin that have different affinities for O₂

question

Post-translation Mod.
Carbohydrate addition

answer

o-glycosylation: OH of ser, thr, tyr
N-glycosylation: NH2 of asn

question

Post-translation Mod.
Lipid Addition

answer

palmitoylation: internal SH of cys
myristoylation: NH of N-terminal gly
Prenylation: SH of cys

question

Post-translation Mod.
Regulation

answer

phosphorylation: OH of ser, thr, tyr
acetylation: NH2 of lys, terminus
ADP-ribosylation: N of arg, gln; S of cys

question

Post-translation Mod.
Modified amino acids

answer

oxidation: pro, lys
carboxylation: glu

question

Secondary structure

answer

alpha helix (right handed) all r groups on the outside?
beta pleated sheet is either antiparallel or parallel (can stack on one another)
beta turn

question

Tertiary Structure

answer

patterns of secondary structure
structural motif->subdomain->domain->fold
1393 folds identifield
no new folds discovered since 2008
nucleotide binding fold, actin fold ect

question

Quaternary Structure

answer

the specific interaction of distinct polypeptide chain subunits (geometrically stoichiometrically)
means to produce fully functional highly stable proteins

question

Structural forms of proteins

answer

globular
fibrous
membrane spanning

question

Factors influencing protein folding

answer

mutations
environment(synthesis, functional site-ph, salt, temp, oxidizers)
chemical modification (enzymatic and non-enzymatic)
protein-protein interactions (chaperones, other interacting proteins, abnormal proteins)

question

Heme

answer

heme porphyrin 9 group found in hemoglobin (4 tetraporphyrin groups)
iron in center (Fe2+)
iron bound by 5 Nitrogens and one oxygen
oxygen pulls heme group into plane with the iron and moves the helix that the heme is attached too (cooperativity)
attached to hemoglobin or myoglobin by proximal histidine

question

myoglobin

answer

a lot of alpha helical structure
single monomer with one heme group binded by proximal histidine
not great at grabbing oxygen nor releasing it
found in muscles in high amounts

question

hemoglobin

answer

has two alpha and two beta subunits (quaternary structure) 4 protomers(monomers)
each protomer has its own heme group
participates in cooperativity(easier to bind with every new oxygen bound)
means multiple affinities for oxygen(sigmoidal curve)
T(tense) state and R(resting) state
if no cooperativity the curve would shift to way right and wouldnt get to 1.0 and not be sigmoid

question

2,3-BPG

answer

allows for hemoglobin cooperativity
called bisphosphoglycerate because PO4 groups on different carbons(interacts with His and Lys
inc BPG a right shift of sigmoid curve happens (takes higher pO2 to get 1/2 O2 sat.)
dec BPG a left shift happens to sigmoid curve(takes lower pO2 to get 1/2 O2 sat.)

question

Fetal Hemoglobin

answer

O2 flows from maternal hemoglobin to fetal hemoglobin
fetal hemoglobin does not bind BPG so it has a leftward shift, so can pick up oxygen more readily from lower O2 environment

question

pH and Hemoglobin Binding Curves

answer

lower the pH (more H+), the more to the right the curve is so it releases its oxygen at high pO2 levels
the higher the pH (less H+) the more to the left the curve so it releases its oxygen at lower pO2 levels

question

Bohr Effect

answer

a decrease in blood pH and increase in CO2 leads to hemoglobin molecules to release there oxygen more readily with the oposite being true
this is causes because an increase in CO2 reacts with water to form carbonic acid, increasing pH
an added proton(H+) from low pH stabilizes the ionic bonds favoring the Tstate of Hb and release oxygen
the high pH removes the H+ from the Hb(in lungs)

question

CO2 transport to Lungs

answer

bulk of it is transported by bicarbonate H₂CO₃
some of it is transported by carbamate of hemoglobin
Hb-N-COO- + H⁺
^|
^H

question

Hb Curve Right Shift

answer

caused by increase in pCO2, temperature, acidity(lower pH) or BPG

question

Carbon Monoxide

answer

binds to iron in heme at a much greater affinity then oxygen
takes long time to come off
if one CO is bound the Hb holds onto its oxygens

question

Detection of Abnormal Hemoglobins

answer

hb genes are expressed co-dominant hence normal and abnormal may be present
a change in AA sequence can result in a change in charge so physical seperation can be used (electrophoresis or chromatography)
can be performed on meltimers or on seperated chains
DNA sequencing can then be performed
common hemoglobinopathies can be quickly detected through simpler techniques like sickle cell preps and blood smears

question

Hemoglobin S and C
statistics

answer

hemoglobin S trait (?A?S) is present in 8% of african americans, and as high as 30% of individuals in some areas of africa and middle east
hemoglobin C trait (?A?C) is present in 5% of african americans

question

6th Amino Acid for
Hemoglobin S and C

answer

A position 6 Glu, isoelectric point is 7.0
S position 6 Val, isoelectric point is 7.2
C position 6 Lys, isoelectric point is 7.4

question

Hb and Alkaline Electrophoresis

answer

carried out at pH of 8.6 because all Hb are negative at this charge and migrate towards anode
the magnitude of the negative charge will determine how rapidly the hemoglobin migrates
many hemoglobin variants migrate to the same point

question

Hb and Acid Electrophoresis

answer

performed in agar with a citrate buffer at pH 6.2 and is a complement to alkaline electrophoresis
agaropectin in the agar binds reversibly to certain AA on the surface or heme pocket
the Hb-agaropectin complex moves based on amount of binding
confirsm S and separates C from other Hb

question

Hb and Isoelectric focusing

answer

many ampholytes(small peptides which carry charge and affect pH) are suspended in agarose
when current is applied these ampholytes establish a pH gradient across the strip
applied hemoglobins migrate to their isoelectric point
this yields sharp bands but more minor bands
static vs dynamic and widely used in pop. screening

question

Hb and High Performance Liquid
chromatography

answer

uses a weak cation column, as ionic strength of eluting buffer increases Hb elute with a particular retention time
Hb with AA substitutions present will have a different retention time that A(2.5 mins)- for example S(4.48 mins) and C (5.15 mins)

question

Hemoglobin S

answer

deoxygenated hb polymerize and sign polymerization leads to formation of helical filaments
Hb S has a greater tendency to polymerize
RBC with S filaments are rigid (sickle) (not elastic)
removed from circ causing an anemia and RBC breakdown products
occlude small vessels causing ischemic injury

question

Hemoglobin C

answer

less soluble then Hb A
tends to crystalize in the cyell
crystal formation is more likely in the oxygenated state
formation of crystals decreases the life span of the RBC
patients with only C Hb have an increased red cell turnover but usually only mild anemia

question

Ischemia

answer

shortage of blood supply to tissues, causing a shortage of oxygen and glucose needed for cellular metabolism

question

Thalassemia

answer

results from an altered expression of one of the globin chains
when there is a significant lack of expression of a globin chain, alternate normal Hb or alternate Hb multimers are present
in beta-thalassemia-Hb A is decreased and Hb A2(delta) and F(gamma) chains are expressed in increased amounts
in alpha-thalassemia- all Hb contain alpha chains so abnormal multimers are present-Hb H and Hb Barts are seen (both real unstable)

question

Structural Hb changes that effect
loading and unloading

answer

FC corner(?94-104) changes affect change configuration movement association with uptake-unloading
C terminus (? 143-146) change affect DPG binding site

question

Polycythemia

answer

increase in hemoglobin levels
can be due to pathologic or physiologic state
pathologic-erythropoietin secreting tumor(or adm of erythropoietin)
physiologic hypoxia-living at high altitude
pathologic hypoxia-chronic lung disease
pathologic failure to release oxygen from hemoglobin

question

Hb Andrew-Minneapolis

answer

lys>Arg substitution in AA 144 in ? globin chain
P50 is 18mm Hg, Loss of 2,3 DPG effect
variant hemoglobin is always heterozygote state(homozygous state is assumed to be lethal

question

Hb Malmo

answer

FG corner Variant-His>Gln AA change in ? chain at position 97
described in southern sweden
does not show up on electrophoresis
increased oxygen binding affinity (does not release at high pO2)

question

1st order kinetics

answer

at substrate concentrations below Km (affinity for enzyme)
reaction rate proportional to substrate
many drugs are metabolized in liver where drug concentrations are below Km but sometimes excessive alcohol ingestion can saturate the alcohol dehydrogenase

question

Zero-Order Kinetics

answer

high substrate concentration reaction
reaction is independent of substrate
all enzyme is in [ES] and not limited by [S], but by the turnover number of the enzyme [Kcat]

question

2nd Order Rate Reaction

answer

dependent on two substrates, substrate and cofactor
thus doubling the concentration of one substrate doubles the rate
while doubling both increases the rate 4-fold
cofactor is either vitamin or coenzyme

question

2nd Order Reaction Enzyme

answer

active site contains unique amino acid domain and cofactor that form covalent bonds with substrate
formation of the transition state requires energy for covalent bond formation
energy derived from ionic, h bonds, hydrophobic, fold free energy of enzyme in ES
once energy barrier is overcome, rx proceeds w/o additional energy

question

Substrate binding site of enzyme

answer

amino acids of the substrate binding site determine what substrate binds
2 models: lock and key model and induced fit model
both models suggest that, upon ubstrate bidning, the enzyme active site changes conformation the the transition state

question

Lock and Key Model

answer

model for substrate binding site of enzyme
amino acid domain of active site determimes the type of substrate bound through hydrophobic, electrostatic(ionic) or hydrogen bonds
ex is glucokinase that binds glucose with ionic bonds and hydrogen bonds

question

Induced fit model

answer

model for substrate binding site of enzyme
substrate binding induces conformational change necessary for hydrophobic, ionic, and hydrophillic bond formation

question

Transition State Complex

answer

substrate activation requires energy for the formation of covalent bond formation of the transition state complex
enzymes increase the rate of reaction,kcat, by decreasing the activation energy through either acid-base or electronic stabilization of the [ES]

question

Transition State Analog Inhibitors

answer

highly potent and selective enzyme inhibitors because they mimic the three dimensional structure of the substrate transition state with pre-formed covalent bonds and thus require no energy of activation
bind enzymes a million fold tighter then substrate, and thus inhibitors can be administered at lower concentrations to avoid adverse drug toxicity
have high affinity(low Km)

question

Serine Protease

answer

cleaves preproteins(zymogens) to make them active
pivotal role in blood coagulation(prothrombin) and digestion(trypsin, chymotrypsin and elastase)
hydrolyze peptide bonds on the carboxyl side of hydrophobic amino acids, Phe, tyr, trp and leu
dissociation of water to hydroxyl group(nucleophile) can attack the protein carbonyl group forming an oxyanion transition state complex
hydroxl addition to carbonyl froms a carboxylic groups, and the proton forms an amine

question

Chymotrypsin Catalysis

answer

catalytic triad(Asp-His-Ser) Asp transfers e- to His nitrogen to initiate acid-base catalysis
removal of serine H+ makes serine oxygen a highly reactive nucleophile, making oxyanion tetrahedral transition state
transition state stabilized by covalent bonds with ser and his functional groups and substrate
acyl enzyme interm. serine covalent catalysis
water donates e- to carbonyl forming 2nd transition state
electron rich tetrahedral draws His H+ to serine
oxyanion base draws the his hydrogen(acid), releases acyl-enzyme and reforms OH serine

question

Thiamine Pyrophosphate (TPP)

answer

activation-transfer coenzyme by forming a covalent bond with substrate(pyravate)
enzyme function amino acid groups rmove a proton from TPP forming a highly reactive carbanion that attacks positively charged carbonyl group carbon causing bond scission

question

Coenzyme A

answer

synthesized from vitamin pantothenic acid functions to add CoA to fatty acids and cholesterol trapping the in cells
in this case instead of bond scission, acyl-CoA synthetase forms a ond btw CoA and fatty acid (acyl) carboxyl group

question

Vitamin Biotin

answer

functions in carboxylase reactions by attaching CO2 to substrate, such as, pyruvate carboxylase converting pyruvate to oxaloacetate
forms covalent bond btw itself and substrate
forms an activation transfer action

question

Pyridoxal Phosphate (Viatamin B6)

answer

reactive aldehyde group binds amino acids and thus is critical for aminotransferase enzyme activity

question

Activation Transfer cofactor features

answer

coenzyme functional group binds enzyme
coenzyme reactive group forms or breaks covalent bond
coenzyme can be thought of as functioning in 2nd order kinetic reactions since they bind enzyme substrate binding site

question

Coenzymes in Oxidation Reduction
Catalysis

answer

co-enzymes NAD+, FAD+, FMN and NADP+ function in the transfer of electron and hydrogen from substrate by donating or accepting eletron as hydride, hydrogen or oxygen
unlike act. transfer coenzymes, oxi-red coenzymes do not form covalent bonds with substrate
lactate dhase binds NAD+ thru AMP moiety and NAD+ functional group accepts hydride ion oxidizing lactate to pyruvate

question

NAD+ and FAD

answer

coenzymes in oxidation reduction catalysis
NAD+ is synthesized from the vitamin Niacin and FMN
FAD is synthesized from Riboflavin
function in the transfer of electron and hydrogen from substrate by donating or accepting electrons

question

Vitamins C and E

answer

function in oxidation-reduction reactions and as antioxidants by donating electrons(reducing agents)

question

Metal Ions
(Enzyme Catalysis)

answer

metal ions have a positive charge and thus function as electrophile(electron loving)
assist in substrate binding to enzyme, stabilization of function groups in acid-base catalysis, accepting and donating electrons in oxidation-reduction reactions
alcohol dhase(ADH) catalyzed oxidation of alcohol to acetaldehyde requires zinc to stabilize serine hydroxyl group that removes a proton from alcohol leaving serine oxyanion stabilized by Zn2+, and thus allowing hydride transfer to NAD+

question

Covalent Enzyme Inhibitors

answer

form tight bonds with function group amino acids of the catalytic site
usually toxins or selective drugs
usually irreversible inhibitors and thus require new enzyme synthesis
examples are aspirin and 5-fluorouracil

question

Aspirin

answer

covalent coenzyme inhibitor
irreversible inhibitor of prostaglandin endoperoxide synthase (PGH)
PGH has endoperoxidase and cycloxygenase activity (COX) and normally abreviated as COX1 and COX2

question

5-Fluorouracil

answer

chemotherapeutic drug
covalent enzyme inhibitor
inhibits thymidylate synthase in the conversion of dUMP to dTMP for DNA synthesis

question

Competitive Inhibitors

answer

do not change Vmax because inhibitor binding is reversible and can be overcome by high substrate concentrations
however Km is increased becasue sub binding impairment
structurally related to normal substrate
product inhibition by products is an important mech of feedback inhibiton: methanol and ethanol competes for alcohol dhase and methanol goes to formaldehyde?
succinate dhase metabolizes succinate to fumarate, which is comp. inhibited by malonate

question

Non-Competitive Enzyme Inhibitors

answer

structurally unrelated to substrate
bind to enzyme, not at catalytic site, to inhibit ES and E
decrease Vmax with out changing Km
cyanide, heav metal ions and some insecticides are all nc inhibitors
Na/k ATPase is inhibited by ouabain
lead forms covalent bonds with sulfhydryl side chain of cystein, causing nc inhibition in things like ferrochelatse

question

Un-competitive Inhibitors
(enzymes)

answer

bind only to the enzyme substrate complex and not the free enzyme
decrease both Vmax and Km
unlike comp inhibitors, uncompetitive inhibitors work best at high substrate concentrations

question

Suicidal or Irreversible inhibitors

answer

irreversible inhibitors bind covalently and only way to overcome inhibition is create new enzyme
also called mixed inhibitors because they bind both E and ES and decrease Vmax and do not change Km
most irreversible inhibitors are environmental toxins or drugs MAO inhibitors allow neurotransmitter to remain active longer
organophasphate (acetylcholinesterase), allopurinol (gout/xanthine oxidase) and aspirin(cyclooxygenase)

question

Transition State Analogs

answer

resemble enzyme transition state substrate
penicillin binds glycopeptidyltransferase, a bacteria enzyme required for cell wall synthesis
partial reaction of penicillin transition state analog with the enzyme results in covalent bond formation and suicide inactivate of enzyme
completely inactive enzyme activity irreversibly

question

Transition State Analog Inhibitors(TSAI)

answer

have been synthesized and are presently used in clinical trials
mimic substrate enzyme transition state and thus do not require energy to form covalent bonds, thus they bind with high specificity and 1 million fold more tightly to enzyme than substrate
high affinity and avidity reduce druge dose reducing adverse effects

question

Alopurinol

answer

TSAI requires covalent bond to inhibit xanthine oxidase and uric acid synthesis from purine nucleosides
allopurinol is converted by xanthine oxidase to its transition state analog oxypurinol that irreversibly inhibits enzyme activity, suicide inactivation

question

Forodesine

answer

inhibits conversion GMP to guanine to
instead converts to GTP and then to dGTP that causes T-cell Apoptosis
inhibits Purine nucleotide phosphorylase(PNP) that functiosn to remove ribose 1-phosphate from purine nucleoside producing the base that can be metabolized to uric acid

question

Oxidoreductase

answer

most common enzymes involved in metabolism that transfers electrons from substrate to co-enzyme NAD+, FAD+, NADP+, or elecrons from coenzyme to the substrate
the enzymes, dehydrogenase, are usually named for the substrate they metabolize

question

Oxygenase

answer

oxido-reductase enzymes
use molecular oxygen as a substrate and normally a co-enzyme for activity

question

Monooxygenase

answer

oxido-reductase enzymes
cytochrome P450
transfer one molecule of oxygen to substrate and other to water
found in reduction of steriods and eliminate of toxic drugs and conversion of drugs

question

Dioxygenase

answer

oxido-reductase enzymes
such as COX enzyme
insert two molecules of oxygen into substrate

question

Oxidase

answer

oxido-reductase enzymes
transfer electrons from the substrate to oxygen producing hydrogen peroxide
peroxidase convert hydrogen peroxide to water, therefore oxidase can have two activities

question

Transferase

answer

catalyze the group transfer reactions
if the transfer group is a high energy group, the enzyme is a kinase
glycosyltransferase transfers carbohydrate while acyltransferase transfers fatty acids
transaminations are important for measuring blood to determine organ damage
synthase also function in transfer of glucose from UDP or ehtanolamine from CDP and unlike synethase it does not require ATP

question

Hydrolase

answer

cleaves C-O, C-N, or C-S bonds by addition of water
most digestion and lysosomal ezymes are hydrolases (chymotrypsin)
hydrolase names indicate the substrate or bonds on which they act, which include petidase, lipases, esterases, and phosphatases

question

Lyase

answer

remove a group nonhydrolytically forming a double bond such as with dhydratase
lyases cleave C-C, C-O, and C-N bond by means other than hydrolysis or oxidation
are many times reversible, creating a new bond and obliterating a double bond
when they favor the formation of a single C-C bond they are often called synthase

question

Isomerase

answer

interconvtert positional, geometric or optical isomers
isomerases that catalyze the movement of a phosphate from one atom to another are called mutases
isomers are molecules with same formula but different structures

question

Ligase Enzyme Catalysis

answer

in contrast to hydrolase, ligase synthesize C-C, C-S, C-O, C-S, bonds by cleavage of a high energy bond
carboxylase addes CO2 to substrate with energy provided by ATP cleavage
Synthetase such as fatty acyl-CoA synthetase attaches CoA to fatty Acid by cleavage of a high energy bond

question

Lineweaver-Burk and Michaelis Menten

answer

michaelis-menten is the graph that shows the Km and Vmax and is all in the second quadrant
lineweaver-burk takes the equation for that and makes it linear, making 1/vmax the y intercept and -1/Km the x intercept

question

Hill Plot

answer

used when a velocity vs substraight curve is sigmoidal in enzyme kinetics
it approximates what a michaelis-menton curve would look like on it, figure out wheres 1/2Vmax is and then keep going over to that point on the sigmoid curve
this point is the S_0.5or K_0.5

question

Monomeric G-Proteins
steps

answer

when GTP is bound the conformation of Gprotein allows it to bind target proteins, which are then activated
gprotein hydrolyzes a phosphate from GTP to form GDP, which changes gprotein confromation and causes it to dissociate from the target protein
GDP is exchanged for GTP, which reactivates the gprotein

question

Glycogen Phosphorylase
and AMP

answer

AMP binds to the allosteric site on glycogen b, making it active and ready to accept two phophate groups, one per catalytic subunit

question

Caspases

answer

internal cellular proteases involved in driving apoptosis(programmed cell death
proteolytic cleavage example

question

Zymogens

answer

class of inactive precursor proteaseswhich prevent them from being active until needed
ex. are firbinogen to fibrin and prothrombin to thrombin
such pre-enzymes are held in reserve until needed and can quickly become enzymes withough gene expression and protein synthesis

question

Regulation of Metabolic Pathways

answer

regulation occurs at the rate-limiting step
feedback regulation
feed forward regulation
tisse isozmes of regulatory proteins
counter regulation of opposing pathways
substrate channeling through compartmentation

question

Assymetry of Plasma Membrane

answer

higher concentration of phosphatidylcholine and sphingomyelin in outer leaflet
higher content of phosphatidylethanolamine and phosphatidylserine in inner leaflet
phosphatidylinositol(cell signaling) is exclusely located in iner leaflet
cis double bonds kink, trans do not(trans fat bad)
flippases transfer lipids between leaflets

question

Lipid Anchored Proteins

answer

third class of membrane proteins
include proteins bound tot he external membrane surface trhough the glycophosphatidylinositolglycan(GPI) anchor, a lipid covalently attached iself to the membrane
uses inositol (which is usually only on the inner leaflet but some cases it is not?)

question

Peroxisomes

answer

single membrane bound that can replicate by by division (no DNA)
oxidative rxs: catalase can degrade H2O2 produced from the first step of beta-oxidation(mit.)
oxidation of very long chain fatty acids(>c20)
conversion of cholesterol to bile acids
originated from ER

question

Peroxisome Defect
Disorders

answer

Zellweger syndrome, neonatal adrenoleukodystrophy, Infantile Refsum Disease
IRD is less severe then the others
Xlinked ALD is defect in ABCD1, the VLCFA transporter to peroxisome(accumulation of VLCFA)(leads to death)
Refsum disease defect in the alpha oxidation enzme, phytanoyl-coa hdase, or PEX7 leading to phytanic acid accumulation (dieting from dairy and ruminant meat can help)

question

Lysosome

answer

single membrane with internal pH of 5.5
membrane proteins protected from digestion y glycosylation
responsible for digestion, elimination of things
orginated from golgi and endosome
lysosomal storage disease is caused by build up of undigested material in lysosome from defect in enzymes (tay-sachs, niemann-pick, gauchers)

question

Mitochondria DNA Genome

answer

encodes 37 genes
13 for subunits of respiratory complexes
22 for mitochondrial tRNA, 2 for rRNA
genetic defects lead to complex disease such as mitochondrial encephalomyopathy
most mitochondria are maternal
MELAS? (possibly a disease)

question

Smooth Endoplasmic Reticulum
(SER)

answer

Microsomes formed by mechanical fragmentation
responsible for lipid synthesis, metabolize drugs and xenobiotich chemicals(cytochromP450) for detoxification, steroid hormone syntehsis
glycogen storage in liver cells

question

Gated Channels

answer

transmembrane proteins form a pore for ions that is either opened or closed in response to stimulus
may be voltage gated, ligand gated, or a regulator change in the intracellular domain(phosphorylation-gated and pressure gated)
Cystic Fibrosis transmembrane conductance regulator(CFTR) is an example of a ligand gated channel controlled by phosphorylation

question

Caveolae mediated transport

answer

potocytosis
cave like structure in membrane
full of sphinoglipids
formed by caveolaen(cholesterol binding protein)
lipid raft?

question

ATP-Binding Cassette
Transporter Superfamily

answer

bind ATP and use the energy to transport hydrophobic molecules across cell membranes
classified based on the homology in the ATP binding domains
consists of 7 mammalian subfamilies based on similarity in gene structure
chart in slides*

question

Fatty Acid Transporters

answer

first fatty acids are freed from dietary fat by gastric lipases and transported across the apical membrane of the intestine actively by FATP4, FAT/CD36, FABPpm, or passively diffuse through lipid bilayer in enterocytes, FABPc facilitates fatty acid transport through the cytosol.
majority is re-esterfied to TAG and excreted into circulation as chylomicrons(CM)
membrane and non-membrane associated transporters

question

Fatty Acid Membrane
Associated Transporters

answer

plasma membrane fatty acid binding protein coopers with CD36 to transport fatty acids
fatty acid translocase/CD36 two transmembrane spanning regions major form responsible forinsuline inducible FA uptake in muscle involved in atherosclerosis
fatty acid transport proteins-six trans transmembrane domains
FATP1(white adipose and muscle), FATP2 and 5(hepatocytes) FATP3(many tissues, FATP4 (enterocytes), FATP6(cardiomyocytes

question

Fatty Acid Non Membrane
Associated TRansporters

answer

cytosolic faty acid binding protein(FABP_c)
facilitate intracellular FA trafficking to targe organelles such as mitochondria or nucleus
serum albumin transports FA to target tissues in blood circulation

question

Glucose Transporters

answer

Na+ dependent cotransporter (Na/K pump helps keep sodium gradient to facilitate this)
facilitative glucose transporter
insulin can stimulate these

question

GLUT1

answer

glucose transporter
in human erythrocyte and blood brain barrier
expressed in cell types with barrier functions
although GLUT1 in erythrocytes has a overall km of 1-7mM, it is abundant to transport serum glucose adequately evin in the hypoglycemic level (2.2mM)

question

GLUT2

answer

glucose transporter
found in liver, kidney and pancreatic ?-cell
high capacity, low-affinity transporter
high Km value of liver GLUT2 allow serum glucose available for other tissues, since liver has its own capacity for glucose production

question

GLUT3

answer

glucose transporter
found in brain(neurons)
major transporter in the central nervous system
exhibits very low Km value for glucose because the brain uses glucose as the only carbohydrate energy source

question

GLUT 4 and 5

answer

glucose transporter
GLUT4 found in adipose tissue and skeletal muscle and is insulin sensitive
GLUT5 is found in intestinal epithellium and spermatozoa and is a fructose transporter

question

Types of Chemical Messengers

answer

Neurotransmitters(nervous system)
hormones(endocrine system)
cytokines(immune system)
retinoids, eicosanoids, growth factors, ect

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Acetylcholine(Ach)

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a neurotransmitter secreted from neurons in response to an electrical stimulus
relays a signal from a motor nerve to a muscle fiber
when action potential is reached, voltage gated Ca2+ channels open triggering fusion of Ach synaptic vesicles with plasma membrane
the release Ach binds to Ach receptors causing its conformational change allowing Na+ to diffuse in and K+ to diffuse out triggering a cellular response
signal is terminated by acetylcholinesterase

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Neuropeptide

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small peptie(4-36 AA) secreted by neurons
neuropeptide alpha-MSH is melanocyte stimulating hormone:anorexigenic peptide (controlling appetite)
neuropeptide Y:released from hypothalmus (orexigenic peptide)

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Eicosanoids

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use gprotein coupled receptors
control cellular function in response to injury
are all derived from arachidonic acid
prostaglandins, thromboxanes and leukotrienes

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Chemical Messangers for
Intracellular Transcription factors

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either hydrophobic or lipophilic
mostly gene-specific transcription factors
transported in blood bound to serum albumin or toa more specific transport protein such as steroid homrone binding globulin(SHBG) or thyroid hormone binding globulin (TBG)

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Cytoplasmic Steroid/thyroid
Hormone Receptors

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asociated with Heat Shock Proteins (HSPs) in absence of ligands (HSP90)
binding of ligands triggers HSP release and migration of the receptor into the nuclease to a specific DNA site called the hormone response element
ex: PR(progesterone), GR(cortisol), MR(aldosterone)

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Nuclear Steroid/Thyroid
Hormone Receptors

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mostly bound to DNA as a heterodimer with RXR in an inactive state without ligands (most like this)
some homodimer or monomer
when active ligand bind, the receptor recruit protein complexes which initiate gene transcription
ex: PPARs(fatty acids), LXR(oxysterol), FXR(bile salts), VDR (vitamin D₂)

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Major Classes of Membrane Receptors

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Ion-Channel Receptors
Receptors that are kinases or that bind kinases
Heptahelical receptors (Gcoupled Protein Receptors)(7membrane spanning alpha helices)

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Tyrosine Kinase Receptors

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Ras and the MAP kinase Pathway
phosphatidylinositol phosphates in signal transduction
JAK-STAT Receptors
Receptor Serine/Threonine Kinases

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Ras and the MAP Kinase Pathway

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a growth factor as ligand for the two proteins of this
receptor dimerizes which activates tyrosine kinase activity
this activity phosphoralates each others receptors
recruits Grb2 contains SH2 domain(recognizes phosphorylated tyrosine residues)
Also recruits SOS contains proline rich domain that binds to SH3 of Grb2 and GEF that associates with RAS
when SOS binds with RAS, RAS exchanges GDP for GTP and becomes activated and goes and activates enzyme Raf (with MAPK)

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Phosphatidylinositol Phosphate
in signal transduction

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signaling molecules can be generated thru tyrosine kinase receptors or GPCR
2 ways to initiate signaling
1:phosphatidylinositol 4, 5 bisphosphate is cleaved to generate 2 important intracellular messengers, diacylglycerol and inositol triphosphate (gprotien uses enzyme PLC)
both calcium from IP3 and DAG activates PKC
2: PI-4,5-bisP is converted by PI 3-kinase to PI-3,4,5-trisP, which serves as a plasma membrane docking site signal transduction protein containing pleckstrin homology domains

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PLC subtypes

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PLC gamma is activated by tyrosine kinase
PLC beta is activated by GPRC

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Phosphatase and Tensin Homolog
(PTEN)

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terminates or inhibits PI3K activation via dephosphorylation of PIP₃ to PIP₂

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PI3 Kinase

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PI3Ks are grouped into 3 classes depending on their structural homology and substrate preference
among 3 classes, class 1 subfam. are involved in PIP3 production and form heterodimers consisting of one catalytic subunit and one regulatory subunit
their regulatory subunits determine the specificity of membrane-receptor association
Class 1A: P85 w/ Rec Tyro Kinase, Class 1B: p101 with GPRC)

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Insulin Receptor

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dimer with each half containing an ? and ? subunit
very divergent signaling pathway
can activate PLC kinase, PLC gamma, actiavte G protein

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Insulin Receptor
PLC kinase pathway

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insulin binds and tyrosine activity phosphorylates each half of the dimers domain
this phosphorylation recruits Insulin Receptor Substrate (IRS)(has an SH2 Domain)
IRS phosphorylated and recruits PI3 kinase(SH2 domain)
PI3 kinase converts PIP2 to PIP3
PIP3 thend does stuff with PDK 1 and PKB which activates and leads to mTORC1 from mTORC2

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JAK-STAT Receptors

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leptin can bind to these
no intrinsic kinase activty
binds the tyrosine kinase JAK(janus kinase)
these phosphorylate each other
they then phosphorylate the JAK-STAT receptor
this recruits STATs that are then phosphorylated
STATs then dissociate and translocate to the nucleus

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Leptin

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regulates neuronal activity of satiety in the hypothalamus to control energy balance and body weight
induces STAT3 homodimer formation
increase in lepten decreases apeteite

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Receptor Serine-Threonine Kinase

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transforming growth factor ? and bone morphogenetic proteins (BMP)
receptor exists as monomer (Type II) but recruits type I receptor after ligand bind
type II phosphorylate serine receptor of type I
this then phosphoryalates serine residues of receptor(R) Smad
this then complex with commom(Co)-Smad and both then translocates to the nucleus

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Smad

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protein found in the receptor serine-threonine kinase pathway
Receptor-Specific Smads: Smad 1,2,3,5, and 9
Common Smad: Smad 4
Inhibitory Smads: Smad 6 and 7

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Phosphatidylinositol Signaling

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activation of phospholipase C by G_?q hydrolyzes PIP2 into DAG and IP3
IP3 stimulates Ca2+ release from the SR
DAG activates Protein Kinase C (PKC)

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Guanylyl Cyclase Receptors

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convert GTP to cGMP
elevated cGMP activate PKG that phosphorylates target proteins
Nitric Oxide Receptor (syn from L arginine)and Atrial natriuretic peptide (ANP) receptor

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Atrial Natriuretic Peptide (ANP) Receptor

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effect guanyly cyclase
membrane bound receptor protein
ANP is hormone secreted by heart muscle to decrease blood pressure and
acts on two sites, kidney and vascular smooth muscle
triggers renal excretion of Na+ and consequently of water to reduce blood volume and relax blood vessels

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cGMP Elevating Drugs

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effect guanylyl cyclase
Glycerol trinitrate: produce NO: for Angina Pectoris
Nesiritide(synthetic ANP): For heart failure
Sildenafil(inhibitor of cGMP phosphodiesterase): Erectile dysfunction

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AMP-Activated Protein Kinase(AMPK)

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A trimeric serine/threonine kinase composed of a catalytic ? subunit and non-catalytic ? and ? subunits
gamma subunit is the AMP receptor(also binds ATP)
AMP biding activates AMPK and ATP binding deactivates it
this is considered an intracellular energy sensor
several upstream kinases can also activate AMPK(LKB1, PLCs, CaMKK?)
effects many metabolic pathways to increase ATP production

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Huntington's Chorea

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progressive motor disability, cognitive and personality decline, and autosomal dominant
onset around 35-50 with death from progression 15-18 yrs later (10% present before 20; usually paternal transmission)
patients usually die from aspiration pneumonia due to difficulty swallowing
caused by presence of mutated Htt gene on chromosome 4
5-35 CAG repeats in normal alle, ;40 high chance disease
shows anticipation(worse with each generation)
no effective therapy
widespread neuronal loss in striatum(initially hyperactive)

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Nucleoside

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nitrogenous base attached to 1' carbon of sugar backbone
becomes nucleotide when a phosphate is added

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Phosphodiester Bond

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between 5' carbon and 3 prime hydroxyl group (uses the phosphate group
creates a 5' end (phosphorus group) and a 3' end (hydroxyl group)

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Forces bringing double helix together

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hydrogen bonding (2 for A-T and 3 for G-C)
the third bond makes the bond lenght shorter for g-c
base stacking
heavily negative charge

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B- form DNA structure
structure

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rise of 3.4 A per base pair
10 base pairs per turn
Major groove and minor groove
right handed usually

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Steps of DNA replication in Euk

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origin of replication identification
unwinding of DNA helix
formation of the replication fork and synthesis of RNA primer
DNA synthesis and elongation
removal of RNA, fill in gaps and link together
reconstitution of chromatin structure

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Proteins and Enzymes involved in
DNA replication

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DNA polymerase
Helicases: processive unwinding of DNA
Topoisomerases: Torsional strain relief
DNA primase: initiates synthesis of RNA primers
DNA ligase: Seals single strand nicks
Single stranded binding proteins: keeps single strands apart during replication

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Pyrophosphatase

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cleaves two of the three phosphates and provides link to the 3' hydroxyl

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Autosomal Dominant

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often seen in multiple generations
mothers and fathers are equally likely to transmit or inherit the disorder
sons and daughters of an affeted parent are equally likely to inherit and transmit the disorder

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Autosomal Recessive Inheritance

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horizontal: usually seen in a single generation of a family
mothers and fathers are equally likely to transmit or inherit the disorder

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Multifactorial Inheritance

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a single trait/phenotype is controlled by multiple genes and/or environment
when plotting array of phenotype you get bellshaped curve (ex is for height:10-15 genes control)
high blood pressure and weight are another ex.
possible threshold for expression is how many u need to manifest?

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Risk estimation for discrete
multifactorial traits

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risk increases with number of affected relatives
risk increases with the severity of the malformation or disease
when something occurs more commonly in one gender, then the offspring of the affected individual of the sex thats less common then higher relative risk
reccurence risk represents average risks and will vary among different families

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pyloric stenosis

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abnormal thickening of the outlet of the stomach
acuses obstruction of gastric emptying and voming
5 times more common in males then in females
this means they have a different threshold
multifactorial disease

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Heritability from twin studies

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h²= (MZ concordance-DZconcordance)/MZ concordance
autism is really high: H²=.95 (this means that the MZ twins got it at a high rate compared to the DZ
helps with multifactorial things

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Types of RNA

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Messenger RNA (mRNA)
ribosomal RNA (rRNA)
Transfer RNA (tRNA): central to protein synthesis as adaptors btw mRNA and aa
Micro RNA (miRNA): regulate gene expression typically by blocking translation or inducing degradation of target mRNAs
Small nuclear RNA(snRNAs):involved in modifications needed to generate mature mRNA

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Nomenclature for describing
sequence variation

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g. = genomic DNA
c.= coding DNA
p. = protein

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Prokaryotic RNA synthesis

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single RNA polymerase
simpler promoter
single factor for initiation
polycistronic mRNA
no RNA processing

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Eukaryotic RNA synthesis

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multiple RNA polymerases
complex promoter regions
multiple transcription factors required for initiation
monocistronic mRNA
extensive RNA processing

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Eukaryotic RNA polymerases

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RNA pol I - for large rRNA
RNA pol II - for mRNA and microRNA
RNA pol III - tRNA and 5s rRNA

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RNA Polymerase I

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found in nucleolus
transcribes the 3 large ribosomal RNAs as one transcript(45S) that is subsequently cleaved: 28S, 18S and 5.8S rRNA
rRNA genes are tandemly linked with many copies per genome
Pol I synthesizes approximately 70-80% of the RNA in a cell

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RNA Polymerase II

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Found in the nucleus
transcribes genes that encode proteins
primary transcripts are subsequently produced into messenger RNA
accounts for approximately 3-5% of RNA in a cell (accounts for least abbundant RNA)

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RNA Polymerase III

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found in the nucleus
transcribes small RNAs, including 5S rRNA and transfer RNA
some pol III transcripts are spliced and/or the RNA sequence is edited after synthesis
transcribes approximately 17-20% of the RNA in a cell

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Eukaryotic promoters

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multiple regulatory sequences
some have a TATA box
other elements too that guide transcription machinary
depends on the gene
might be enhancer elements

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Termination sequences
for transcription

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one sequence causes a hairpin loop
second is rho factor that sits on a sequence near end and moves along and when it catches up to RNA polymerase it terminates it

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Polycistronic mRNA

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found in prokaryotes
RNA polymerase transcribes multiple proteins
usually called an operon and all the genes trancribe proteins that usually work together
an example is the lactose operon

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Assembly of Basic
Transcription Apparatus

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TFII D, along with coactivators and TATA binding protein, binds to promoter
TFII A and B then recruited
RNA polymerase then sits down next to TFII B
TFII F, E, and H then bind to the complex

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Generation of mature mRNA

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capping: serves to seal 5' end of mRNA and decrease rate of degeneration, serves as recognition site for ribosome
poly (A) tail
intron splicing

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Synthesis of Poly-A Tail

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polyadenylation signal (AAUAAA) upstream from the termination site
poly(A) polymerase uses ATP to add a poly (A) tail

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Intron Removal

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U1 binds/ cuts splice donor site (GU)
U2 binds lariate aceptor site(middle of intron) (Adenine)
U4, 5, 6 join complex and link to U2
U 5 identifiies splice acceptor site
intron is cut away as a lariat and the two exons are ligated perfectly
elements of splicesome complex are reusable and intron lariant is rapidly degraded

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Processing of tRNA in nucleus

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introns are first removed (unique and doesnt involve splicesome
then modification of bases and addition of CAA at the 3' end (site of AA. attachment

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Structure of tRNA

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3 loops: dihydrouridine(D) loop, Ribothymidine (T) loop (T-C loop), and anticodon loop that recognizes
70-95 nucleotides in length
a.a. attached to the 3' most base
the T-C loop is involved in ribosome binding
anticodon base pairs with the mRNA codon (antiparallel)

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RISC Complex

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formed from the dicer complex that is fromed from pre-miRNA after it leaves the nucleus
this can either cause mRNA cleavage or translation repression

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Wobble hypothesis

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allows for not perfect match
anticodon C binds with codon G
anticodon A binds with codon U
anticodon U binds with codon A or G
anticodon G binds with codon U or C
anticodon I binds with codon U, C, or A

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Point Mutation

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silent: change that specifies the same A.A.
missense: change that specifies a different A.A
nonsense: change that produces a stop codon

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Insertion Mutation

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causes frameshift or new AA
addition of one or more bases
ex is CAG inserts Gln(polyQ), huntington's disease

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Deletion Mutation

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frameshift or loss of AA
loss of one or more bases
example is ?-Thalassemia due to frameshift

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Requirements

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mRNA
Aminoacyl-tRNAs and aminoacyl tRNA synthetases
Ribosomes - "ribozymes"
Eukaryotic initiation factors (eIFs; G-proteins)
Elongation factors EF1A and EF2 (G-proteins)
Energy in form of ATP and GTP

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Formation of Aminoacyl-tRNA's

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each AA has a corresponding aminoacyl-tRNA synthetase
only 1 tRNA which recognizes the AUG start codon: methionyl-tRNA_i^Met
charging a tRNA requires energy in the form of ATP and is a two step process

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Combing of an AA to tRNA

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first aminoacyl tRNA synthetase (enzyme) attaches to an amino acid using ATP
this creates a Enzyme-aminoacyl-AMP complex
it then addes the AA to a tRNA and the AMP and enzyme dissociate
atRNA synthetase recognizes three sequences( on d loop, anticodon and 3' end) in order to attach right AA to right tRNA

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Three Steps of Translation

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Initiation: binding of initiator tRNA to initiator codon at P site of ribosome
Elongation: binding of aminoacyl-tRNA to A site; formation of a peptide bond; translocation; ejection of spent tRNA
termination: release of completed peptide

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Euk. Translation Initiation
Requirements

answer

capped mRNA
small 40S ribosomal subunit
eIFs (eIF2 with GTP binds initially to tRNA)
initiation Met-tRNA: complex uses ATP to scan the mRNA for Kozak AUG start sites (eIF4F helps here); GTP (on eIF) is then hydrolyzed and eIFs are release
large 60S subunit binds to complete the ribosome
eIF3 had been stoping large subunit from initially binding

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Eukaryotic Initiation of Translation

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cap at 5' end of mRNA binds eIF4F and 40S subunit with met-tRNA
mRNA scanned for AUG within a kozak consensus sequence
12 or more eIF
80S ribosomes (60 and 40 subunits)
First AA is methionine

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Prokaryotic Initiation of Translation

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mRNA has no 5' cap
Shine-Dalgarno sequence upstream of AUG binds to complementary sequence of 16S rRNA
3 IF
70S ribosome (50S+30S)
first AA is formyl-methionine

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Translation Elongation

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eEF1A +GTP + tRNA attach to A site and GTP is hydrolyzed
RNA then catalyzes the peptide bond formation(peptidyl transferase)
eEF2+GTP then move the tRNA with AA chain from A to P site (translocation)
tRNA that was in P site moves to E site and is ejected, along with eEF2 and GTP is hydrolyzed

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eEF1B

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helps in the recycling of eEF1A in translation
eEF1B binds GTP
it then exchanges the GTP for the GDP bound to eEF1A after it had went through a cylce of translation
makes translation go much faster

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Termination of Translation

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no tRNAs pair with STOP codons
protein release factors (RFs) bind to stop codons: RF-1 binds UAA and UAG, and RF-2 binds UAA and UGA
binding of RFs cause peptidyltransferase to hydrolyze the peptide-tRNA linkage and the polypetide is release
tRNA is released from P site and the two ribosomal subunits separate as a result of GTP hydrolysis

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Energy Requirements for Translation

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multiple high energy bonds required for each peptide bond made: 2 ATP for charging aminoacyl tRNA, 1 GTP is used to bind tRNA to A site, and 1 GTP to translocate tRNA peptide from A to P site
Energy requirements at initiation and termination: 2 ATPs for charging Met-tRNA, ATP needed for helicase to undwind hairpins/loops in mRNA structure
1GTP to form initiation complex at AUG
1 GTP for termination associated with dissociation of the ribosomal subunits

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Inhibition of Translation- Antibiotics

answer

streptomycin: inhibits initiation and causes misreading of the RNA
tetracycline: binds small (30S) subunit and inhibits binding of aminoacyl-tRNA
chloramphenicol: inhibits peptidyl transferase activity of the large subunit (50S)
erythromycin: binds reversibly to large subunit(50S) and inhibits translocation/peptidyl transferase

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Targeting sequences in the Polypeptide

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secretory pathway-N-Terminal signal peptides
ER
membrane insertion sequences-hydrophobic helices
nuclear localization sequences
mitochondrial import sequences
peroxisome signals

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Post translational modifications
(proteins)

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glycosylation-golgi, lysosomal signals
proteolytic cleavage sites
phosphorylation
lipidation

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Core Sugar Complex

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formed in the lumen of the endoplasmic reticulum
formed from seqential addition of sugar residues at non-reducing end of dolichol phosphate
this core sugar can be added to a protein, and influences the protein folding (hydrophillic)

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Mannose Phosphate

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can be added to an protein
mannose-P receptors bind it and a vessicle is formed that transport the protein to the lysosome

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Types of DNA binding motif

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assist in transcription factors bind to DNA
Zinc fingers
leucine zipper
helix-turn-helix
helix-loop-helix

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Zinc Finger Motif

answer

require a chelating agent? (Zinc)
must chelate either histidine or cysteine to stabilize the finger motif
recognize specifc sequence by grabbing the DNA
NRS(alpha helix) recognizes specific DNA sequence on major groove of DNA

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Leucine Zipper
****

answer

7 hydrophobic luecines are alligned on one side of the protein and the other side are polypeptides
bond using hydrophobic contraction
usually homo or heterodimer transcription factors
arginine and lysine positive amino acid can bind to something in major groove??

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Helix-Turn-Helix
******

answer

monomer??
first helix recognizes DNA major grove
2nd and 3rd stabilize DNA binding
first helix is like luecine zipper and has a polypeptide that recognizes major groove using positive charged arginine or lysine

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DNA Response Elements

answer

direct repeat: AGGTCAnAGGTCA - called DR1
add more "n" to make DR2, DR4, ect
palindromes are either inverted(AGAACA(n) TGTTCT) or everted: TGACCT(n) AGGTCA)

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Ligand Dependent Transcription factor Activation
(nuclear receptors)

answer

LBD; 12 aplha-helices(H1-H12) and 2 beta turns are arranged in three layers
a few helices (H3, 5, 10, 11) form a ligand binding pocket that is struturallly conserved hydrophobic surface for ligand interaction
H12 in the AF-2 domain hangs out the ligand binding pocket like a waving arm, blocking coactivator recruitment
upon ligand binding, conformation chages cause H12 to reposition and allow AF-2 to be functional to recruit coactivators via LxxLL NR box
in process, H12 encloses the binding pocket and forms additional H-bond with ligand to stabilize the conformation

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Steroid receptors
****

answer

nuclear receptor
recognize AGAACA direct sequence
found in cytosol
ER recognizes AGGTCA
GR(glucocorticoid), MR(mineralcorticoid), PR (progesterone), AR(androgen), ER(estrogen)

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RXR heterodimers
***

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nuclear receptor
AGGTCA is recognition sequence
TR(thyroid): DR-4 , VDR(vitamin D3): DR3, PPAR?(fatty acids): DR1, FXR(bile acids): IR1, LXR(oxysterols): DR4, RAR(retinoic acid): DR1
TR, VDR and RAR occupy 3' end and RXR the 5' end
PPAR, FXR recognize 5' site and RXR at 3'

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Class II and III
(thyroid hormone receptor)
*****

answer

thyroid hormone receptor always in nucleus
T3 recognizes the TR, this then recruits coactivator
when ligand not present, the co-repressor complex containing HDAC will be present

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Class I Receptor
*****

answer

Glucocorticoid receptor
in cytosol and usually associated with HSP(Heat shock protein)
when ligand binds, HSP dissociates and GR receptor moves to nucleus

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Peroxisome Proliferator Activated Receptor
(PPAR?)
******

answer

normally expressed in liver
invaolved in beta oxdiation
ligands are polyunsaturated fatty acid(PUFA)
also involved in anti-inflamatory function
drug ligands are fibrate drugs, cofibrate, benzafibrate, fenofibrate
recognizes DR1: AGGTCA
targets acyl CoA synthetase, CPT1, and reduces ApoCIII

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Peroxisome Proliferator Activated Receptor
(PPAR?)
*****

answer

ligand is polyunsaturated fatty acid (PUFA)
expressed in adipose tissue
drugs are TZD avandia
target lipoprotein lipase, SCD1, and GLUT4
decreases serum glucose and FA, increaes insulin sensitivity, increase number of adipocytes, results in weight gain
target for antidiabetic drugs
oposite from PPAR alpha

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Peroxisome Proliferator Activated Receptor
(PPAR?)
*****

answer

highly expressed in muscle
ligands are polyunsaturated fatty acids(PUFA)
switch type II(fast and glycolytic) muscle fiber to type I fiber (slow and oxidative phosphorylation)
increases endurance with exercise
target genes are CPT!, uncoupling protein, lipoprotein lipase

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cAMP Responsive Element Binding Protein
(CREB)

answer

basic luicine zipper transcription factor that binds to palomdromic sequence as homodimer
regulates gluconeogensis
Glucagon binds to Gprotein receptor wihc activatys AC which creates cAMP which activates PKA
PKA activates CREB activates PEPCK(major gluconeogenic gene) by recruiting CBP and TORC2 coactivators

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Metformin

answer

anti diabetic drug
blocks CREB activation(hepatic gluconeogenesis)
activates LKB which activates AMPK
AMPK phosphorylates TORC2 which causes it to leave the nucleus, where it is supposed to coactivate PEPK with CREB

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Foxhead transcription factor
(FOX proteins)
*****

answer

involved in insulin signaling
winged helix TR that bind as monomer or dimer
Insulin normally activates IRS2 which activates AKT which phosphorylates Fox01 which causes it to leave the nucleus
Fox01 normally binds to IRE and promotes PEPCK genes
FoxA2 regulates FA oxidation and ketogenesis during fasting and it is sensitive to IRS1 and 2
insulin resistance decreases IRS2 signaling(hyperinsulin anemia)-Fox1 would be in nucleus and FoxA2 in cytosol

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Sterol Responsive Element Binding Protein
(SREBP1 and SREBP2)
*****

answer

important for lipid and cholesterol regulation
basic helix-loop-helix leucine zipper trans. factor
in ER mebrane as associated with SCAP and INSIG(er protein especially)
when cholesterol is low they translocate to Golgi where S1P and S2P cleave SREBP and release active form of SREBP-then moves to nucleus and promotes SRE gene transcription
SREBP2: cholesterol synthesis and uptake
SREB1: fatty acid synthesis:
both found frequently in liver?

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Telomerase

answer

when DNA is being replicated there is a 3' overhang on the leading strand. this is a telomeric sequence
a telomerase binds here and protects it from being chewed away
makes a long telomerase sequence end that wont matter if it is chewed away

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DNA Repair Systems

answer

must be able to recognize the wrong base or bases
must be able to remove the wrong base or bases
must repair the newly made gap with a DNA polymerase and DNA ligase activity
mismatch repair(copy errors, single base or up to 5), base excision repair(env. chemical radiation, spontaneous degradation), nucleotide excision repair(chemical or radiation damage to a DNA segment), double strand break repair(ionizing radiation, chemotherapy, oxidative free radicals)

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Werner Syndrome

answer

is the gene on the 8th chromosome that codes for a DNA helicase involved in many DNA repair and processing pathways
helicase may also have a role in telomere maintenance
no known conventional therapy for this genetic defect
autosomal recessive

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Regulation of operons by repression

answer

operon normally inhibited by a represser at the operator sequence upstream from the structural genes
an inducer (lactose) can come in and bind with the repressor, thus inactivating it
this allows for RNA polymerase to bind

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Lactose Operon

answer

allolactose binds to repressor, inhibiting it
in presence of lactose and glucose, the genes are on but not transcribing very fast
when glucose is low and theres lactose, cAMP activates CRP that binds to the promoter
this speeds up transcription

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Eukaryotic Gene Expression
Regulation

answer

initiation of transcription
RNA processing(cap and poly A addition)
RNA splicing and alternative splicing
transport of mRNA from the nucleus to the cytoplasm
regulation of mRNA stability-microRNA(miRNA)
gene amplification
gene rearrangement

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Chromatin Remodeling

answer

modifiers such as acetyltransferases can promote or deter the activity of more substantial chromatin remodelers
chromatin remodelers organize the nucleosomes to promote gene activation or repression
such remodelers may either slide or eject necleosomes to promote DNA access to other transcription factors
it transfers a neutral acetyl group to a positive N of lysine, this causes the bond between histone protein and DNA to lessen because it was postive to the DNA negative before

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Phases of the Cell Cycle

answer

G1: 6-12 hours
S phase(DNA replication): 6-8 hours
G2: 3-4 hours
M (mitosis): 1 hour

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Cyclin binding

answer

cyclin binds to CDK, moving the blocking group
this allows a threonine to become phosphorylated
this then activates the CDK

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CDKs and the cell cycle

answer

G1 up to restriction point is coordinated by CDK4/6 that is activated by Cyclin D (allows cell to go past restriction point)
After restiction point CDK2 and Cyclin E push the cell into S phase
CDK2 and cyclin A then push the cell through S phase to G2 phase
CDK1 and cyclin A/B pushes cell through G2 phase
CDK1 and cyclin A/B also push cell through mitosis

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Cell Cycle Check Points

answer

surveillance points which help ensure the mother cell passes accurate copies of its genome to the two daughter cells
is the cell ready for division?, does the replicated genome contain errors, or is damaged?, Is DNA synthesis complete?, Are chromatids properly assembled on mitotic spindle?

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Stop Sign/Restriction Point

answer

first a growth factor initiations a cellular pathway(Ras/Raf)
eventually Cyclin D is produced and it binds to CDK4
this phosphorylates retinol blastoma protein(Rb) which is a tumor supressor and inhibitor
this inactivates it, freeing up E2F
E2F then activates a massive series of genes to progess the cell cycle
CKI can block the CDKs from working
Cylin E and CDK2 can also help in inactiving the Rb

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CKI's Cyclin Kinase Inhibitors

answer

two classes: those that inhibit CDKs 1, 2, 4, 6 (p21) and those that only inhibit CDK 4 and CDK 6
these are proteins that directly bind to the cyclin-CDK compleses to inhibit CDK activity
p21 has an important role in stopping the cell cycle when DNA damage occurs

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p53

answer

checkpoint protein(tumors can result from mutation)
when it is induced:
signals that theres damage in DNA, so it induces p21
if it doesnt get fixed, it induces apoptosis (activates caspases)

question

Steps of Mitosis

answer

prophase
prometaphase
metaphase
anaphase
telophase

question

Mitosis Simplified
Early

answer

abrubt increas in M-CDK(cyclin B) activity drives early mitosis events(chromosome condensation and alignment
active CDK1 activates proteins that lead to the assembly of mitotoic spindles and chromatid attachment

question

Mitosis Simplified
Late

answer

anaphase promoting comples(APC/C) helps drive separation of the sister chromatids
anaphase promoting complex(APC/C) degrades cyclins also degrades other anaphase inhibitory proteins

question

Major Classes of Signals
for Eukaryotic Cell Growth

answer

mitogens(activate cell cycle)
cell growth factors (increase cell size, promote protein synthesis)
survival factors (mainly inhibitors of apoptosis)
all three of these are operating in cancer cells

question

rampamycin

answer

used for transplants because it is an immunosupresnet
inhibits mTOR?

question

mTOR
basics of activation

answer

integrator of nutrients signals to cell growth
rampamycin, mitogens, amino acids, energy and DNA damage effect it to do other thing
must be activated during process of growth
serine-threonine kinase
after activated it finds targets to phosphorylate
some of its effects are cell migration, cell cycle progression, cell growth/size, cell survival, autophagy
can be activated by a G-protein that is dependent on low levels of AMP
GPrtotein also activated by AKT that is activated by insulin
mTOR activation signals to allow ribosome translation***

question

mTOR

answer

TOR enzyme complex is a central controller for cell growth(accumulation of mass
metazoan(multi tissue) TOR is essential for growth during early development(deletion of the TOR gene is embryonic lethal in mammals)
TOR inclunce in mature or aging organisms is partly negative: unwanted cell proliferation; continued growth without division may lead to protein aggregates and abnormal proteins that may induce late life malfunction

question

S6K1

answer

activated by mTORC1
this then can then activate lipid biogenesis, metabolism, translation, cell size regulation, DNA replication, RNA processing
also takes part in a negative feedback mech by inhibiting insulin receptor substrate
mTORC1 can also activate Grb10 that inhibits the insulin like growth factor receptor

question

Medical Genetics

answer

among the 5000 listed genetic diseases, over 1100 genes can be tested clinically
half of the 2% of fetuses with major genetic abnormalities have abnormal chromosomes
30% of children admitted to the hosphital have a genetically related problems

question

Centromere Location

answer

metacentric: when centromere is in the center
submetacentric: when centromere is closer to one side then the other (but both still the same length)
acrocentric: when the centromere is at one end and one is sticks out a little farther then the other one
p arm is the short arm, q arm is the long arm

question

Banding patterns

answer

can depend on different ratios of the basepairs
if stained with an A-T binding protein then parts of a chromosome would look brighter if it had A-T and dimmer if it had G-C
when fluorescing basepairs, the more in a row, the more exponentially bright it is(2 - 4x brightness, 3- 9x brightness)

question

Acrocentric chromosomes

answer

contain stalks and satellites
about 10 chromosomes like this
ribosomal genes tend to be on stalks

question

Down Syndrome from Trisomy 21

answer

palpebral fissures are slanted upward and outward
flat nasal bridge
round face
small mouth, thick lips, often large tongue
average IQ of 50 at 5 years, 38 at 15 years and later degenrates further
hypotonic infants (floppy)

question

Nondirective counseling

answer

provides all known information and allows the patient to decide
couple informed of fetal status, advised of the possibilities, and then advised to decide without the help of a counselor or physician but with trusted individuals and families with similarly affected patients

question

Molecular Cytogenetics

answer

can test chromosome copy number and single gene copy number
enabled by cloned genes and other DNA fragments that can be sequenced, labeled with specific fluorescent colors, and hydridized specifically to similar or identical DNA sequences on chromosome bands, and viewed at 1000-fold magnification

question

Microarray Principles

answer

microarrays test hundres to many thousands of specific chromosome targets simultaneously by labeling total DNA from a patient sample in the first color and total DNA from normal DNA in the second color and hybridizing these to all of the specific cloned genomic sites bound to previously designated locations on the microarray
comparison of both fluorescent colored signals at each location determines whether the patients DNA has less, the same, or more copies of the DNA target than normal DNA. many locations to small to be seen on banding or FISH can be tested at the same time to look for abnormal gene copy numbers in doezens of gene disorders as well as abnormal chromosome locations
FISH is the clinically validated test used to confirm abnormal microarray results

question

Retinoblastoma

answer

may be inherited or arise following two gene mutations to both retinoblastoma genes in the same cell
used to pluck out the eye
looks like white spot in eye
prenatal diagnosis, routine eye exam
laser beam destroys early tumors
inherit one bad gene, and mutation causes other, or two bad mutations

question

Other approaches to
Treating genetic diseases

answer

3. transplantation(adult polycystic kidney disease)
4. modifying diet (maternal PKU)
5. Gene therapy(adenosine deaminase)

question

Enabling PCR Attributes

answer

primer selection: four different basepair possibilities at each of 17 locations
temperature resistant polymerase from bacteria growing in hot springs allows repeated cycling
logarithmic amplification of target sequence

question

Chorionic Villus Sampling(CVS)/ Maternal Decidual Contamination

answer

used to test placenta
tests for circulating fetal DNA in maternal circulation
one type of error though is there could be abnormal cells in circulation but the fetus is actually normal

question

X-Inactivation

answer

occurs after fertilization at the 64-128 cell stage of the blastocyst(embryo) and inactivates about 80% of the genes
results in turning off the genes during the remainder of the life cycle prior to gametogenesis or fertilization. Methylation of the 5' (upstream) promoter region of the genes is involved
the XIST(Xinactivation gene) in proximal long arm initiates inactivation
turns off about 70% of the genes on average

question

Perutz Nobel Prize 1962

answer

hemoglobin protein conformation

question

Gene Deletion (decreased)
Diseases

answer

PMP22: hereditary neuropathy with pressure palsy
SNRPN: Prader-Willi Syndrome
UBE3A: Angelman Syndrome (adjacent to SNRPN)

question

Gene Duplication(increase)
Diseases

answer

PMP22: Charcot-MArie-Tooth Type IA
SNRPN: Autism

question

Imprinting and Uniparental Disomy(decearsed)
Diseases

answer

SNRPN: Prader-Willi Syndrome
UBE3A: Angelman Syndrom (adjacent to SNRPN)

question

Diagnostic Criteria for Autism

answer

severe abnormality of reciprocal social relatedness(plays alone, dislikes being held)
severe abnormality of communication skills(incl. laguage)
restricted, repetitive behavior, interests, activities, and imagination
early onset (before age 5)
can be caused by extra genes upstream?

question

UPD-Uniparental Disomy

answer

starts out with three copies of a chromosome at fertilization
one needs to be lost: if the parent that gave two's chromosome is lost it would be a normal individual
If one from the father is lost then two from a single parent (1/15000)
gene turning off??

question

Reported Abnormal Uniparental Disomy

answer

Parental and Maternal: Chromosomes 14 and 15
Paternal: Chromosomes 1, 2, 6, 7, 11(lethal)
Maternal: chromosomes 9, 16, 20, 22
Suggested: chromosomes 4, 5, 13, 21, X

question

Micro Array

answer

used to define deletions and duplications of large regions of a genome
use chips with attached segments of genetic material
have test DNA and reference DNA
if they both bind to same site, mix of color
ratio of test dna/reference is elevated then evidence of duplication
if ratio is under represented then evidence of deletion

question

Classic Sanger Sequencing

answer

took segment of DNA and chopped up into parts with nucleases
PCR amplified it
add in nucletides (some labled didioxynucletides)
created random length segments and sized them on gel
in direction of larger size you can read the sequence
would take a long time
new method is similar to the old but uses computers and lasers to sequence after multiplying and matching fragments in parallel

question

Allelic Heterozygosity

answer

different mutations in same locus
a disease can be more or less severe depending on where in the allele the mutation happens
severity caussd by the difference in the particular alleles

question

Locus Heterozygosity

answer

multiple genes actually contribute to same phenotypic trait
classic example is retinitis pignitosum: can be due to any one of 15 gene mutations (several autosomal dominant, recessive and x linked)

question

Variable Expression within a family

answer

both mother and son can have an autosomal dominant defect but mother is much less severe
this could be due to modifying genes-one or more independent gene or sequence involved in the expression of a phenotype

question

Penetrance

answer

extreme example of variable expression
gene is present but not manifest
modifying genes work in such a magnitude that you cant even detect the problem in the one not manifesting it
equation is number of mutations with phenotype divided by number of individuals with mutation x 100
complete penetrance is when it = 100% and incomplete is anything less

question

Delayed Onset Dominant Conditions

answer

example is poly cystic kidney disease
results from one heritable mutation and one somatic mutation
the somatic mutation in ADPKD would be inside kidney cystic cells and the autosomal would be in all cells

question

Imprinting

answer

matters which parent passes on a mutation in an imprinted gene
if the gene is inactivated in the mother and active in the father-then it will only be manifest if the mutant gene is inherited from the father
imprinted is erased and re-established with the production of gametes

question

Considerations about use
of Recombinant DNA

answer

must also include appropriate promotor when inserting gene
if bacterial system used, the inserted DNA has to be of viral or bacterial orgigin or the cDNA sequence from eukaryotic genes(no introns)
yeast have advantage of being euk. so can process pro-mRNA
simple peptide products are made. if the peptide requires further modification, it likely has to be performed separately
in most cases the producing cell has to be disrupted and the product purified

question

Products produced by recombinant
DNA technology

answer

vaccines-hep B
peptide hormones-insulin, prathyroid hormone, growth hormone
bone marrow stimulating factors-erythropoietin, GM-CSF, G-CSF
coagulation factors-factor VIII, factor IX and tissue plasminogen activator
interferons and several interlukins

question

Treatment of Human Genetic Disease

answer

symptomatic treatement is most common
replacement therapy
compensation for functional deficits using drugs
use of small molecules as salvage agents
transplantation (provide stem cells from normal donor, risk of conditioning treatment and GVHD)
DNA transfer therapy(gene therapy)

question

Transplantation

answer

used in a number of cases: bone marrow for Hg SS, Liver for some MPS, kidney for familial FSGS, polycystic kidney
issue of ability to find transplant and risks of longstanding immunosuppression
natural extension would be to transplant manipulated stem cells derived from patient

question

Issues with gene therapy

answer

delivery of the material and place of delivery(transfection, direct insertion
sustained expression - stem cell vs somatic cell
avoidance of deleterious immunologic or tissue based responses
potential for inducing secondary disease based on DNA disruption by DNA insertion

question

Epigenetics (modern definition)

answer

modifications of DNA or associated proteins that are not base pair sequence variations that carry information from mother cell to daughter cells
these modifications result in a heritable phenotype without changes in the DNA sequence

question

types of epigenetic information

answer

modification of chromatin structure (CTCF)
chromatin factors(trithorax proteins, polycomb group proteins)
histone modification: methylation, acetylations, ubiquitinylation, and phosphorylation
methylation of DNA

question

DNMT1 and DNMT3A

answer

DNA methyltransferases
recognize the CpG in DNA and then transfers methyl group to it
series of enzymes that can later remove this methylation (TETs)

question

Imprinted Genes

answer

methylated genes that keep there methylation during fertilization
fair amount on fathers genes and less so on mothers
imprinted dont change during genome wide demethylation (De nevo genome wide methyl.)
homologs from mother are expressing so only hers effect body when fathers are imprinted
violates usual rule of inheritance(usually both alleles in a heterozygote are equally expressed)
cells that are destined to make gamates lack all imprinting

question

Hyper vs Hypomethylation Responses

answer

if one is hypermethylated, they would have a lack of response to an environmental stimulus
if they are hypomethylated they would have a heightened response to the same stimulus
cancer tends to come from cyclin dependent kinase inhibitor (CKI's) genes being hypermethylated

question

Genomic Imprinting Disorders

answer

Prader-Willi syndrome (fathers gene is mutated)
Angelman syndrome (mothers gene is mutated)
both on chromosome 15qII
mothers gene is shut off already in prader
fathers gene shut off already in angelman

question

Prader-Willi Syndrome
Inheritance

answer

Usually caused by micro deletion in region q11-13 of the paternally transmitted chromosome 15
several genes in this region are genomically imprinted in the maternal chromosome(turned off)
hence, if there is a paternal deletion in this region, there are no active genes and you get the syndrome
mental retardation, hypotonia, uncontrolable appetite, paternal chromosome has deletion

question

Angelman Syndrom
Inheritance

answer

Usually caused by micro deletion in region q11-13 of the maternally transmitted chromosome 15
several genes in this region are genomically imprinted in the paternal chromosome(turned off)
hence, if there is a maternal deletion in this region, there are no active genes and you get the syndrome
microcephaly, severe mental retardation with no speech, and jerky ataxic movements; maternal chromosone has a deletion

question

UBEA3A Gene

answer

produces a critical ubiquitin ligase enzyme
requires the maternal allele to be expressed in brain during development
Angelman syndrom involves a deletion of this gene in the maternal genome

question

Genetic Testing for Medical Care

answer

establish the diagnosis in a symptomatic person
predictive testing for a relative at risk for a treatable disorder (early treatment, surveilance for a treatable manifestations that appear over time)

question

Genetic Testing For Personal
Decision-making

answer

predictive testing for a relative at risk for an untreatable disorder
carrier testing(autosomal recessive or x linked recessive)
prenatal diagnosis/preimplantation genetic diagnosis (PGD)

question

Phenotype First Approach
(genetic testing)

answer

clinician uses detailed phenotypic findings to determine gene(s) most likely to be mutated
clinician selects test method(s) most likely to detect a mutation
mutation screen including most common known mutations, sequence analysis, deletion/duplication testing(MPLA, qPCR)

question

Spinal Cerebellar Atrophy, type 3
(SCA3)

answer

autosomal dominant disease that results from expansion of a CAG trinucleotide in the ATXN3 gene
100% of cases can be detected by assessing the CAG Repeat number
ataxic gait, sensory deficit, speech problems, tongue fasiculations, pyramidal signs

question

Tay-Sach's Disease

answer

autosomal recessive condition
neuronal storage disease
a mutation of HEX A gene resulting in nonfunctional hexosaminidase A
six mutations that have a 98% chance of being detected

question

Bayes Theorem

answer

multiple independent determinations of risk can be combined to result ina much refined risk extimate
start with the prior(baseline) probability
multiply this with conditional probabilites determined by biochemical or genetic testing-arrive at joint prob.
can then determine a posterior(final) prob by dividing the joint prob. of the condition of interest by the sum of all joint prob.

question

Presymptomatic testing

answer

testing where if you find a mutation, you will develop the disease
an example is huntingtons disease
if you dont have the mutation, you will not develop symptoms of the disorder
issues include effect of knowing that the condition will develop and consequences for employment and health insurance

question

Predispositional Testing

answer

if mutation is found you have a higher risk of developing the disorder but not 100%
if the mutation is not found you have a lower risk of developing the disorder but not 0%
issues are ability to modify outcome, stress of uncertainty and possible discrimination

question

Genotype First Aproach
(genetic testing)

answer

often used for broad phenotypes(ex develop delay, multiple anomalies)
use multi-gene panals (genes included and test methods vary by lab)
whole exome sequencing (WES): sequencing of all coding regions = ? 1% of genome
CGH based array methods

question

Problems with Genotype First
Genetic Testiing

answer

may not find anything, what is detection rate?
more genes tested, more variants of unkown significance
patients phenotype may not fit well with what is known about the disorder at the time

question

Prenatal Diagnosis

answer

sample-parental samples, fetal DNA in maternal serum, chorionic villus sampling or amniocentesis, IVF with blastomere biopsies
Testing-FISH and/or karytype, limited or whole genome array CGH: single gene, gene panal or exome sequencing
issues: timing(only 20wks from finding out), significance of results, collateral effects(ultrasound might detect structurally abnormal fetus), cost

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