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Biochemistry• Molecular Biology – nucleic acid + protein
structure/processing
• Cellular Biology – organelle structure/function, basic cellular processes
• Nutrition/Metabolism – catabolic/anabolic pathways, metabolic disorders
• Laboratory Techniques
• Genetics – pedigree analysis, disease inheritance
• Basic nucleic acid biology • Energy metabolism• Cell cycle• Genetics, pedigree analysis• Sample questions
Nucleic Acid Biology I• Purines (A,G) = 2 rings
• Pyrimidines (C,T,U) = 1 ring
• G-C *3 H-bonds
• A-T *2 H-bonds
• Cytosine deamination uracil• *Base excision repair (why?)
Types of mutations:• Silent = same amino acid
• Degenerate code, tRNA wobble
• Missense = different amino acid (severity of mutation will vary)
• Nonsense = Early stop codon (worst scenario, if truncation occurs early in sequence)
• Frameshift = addition or deletion that is NOT a multiple of 3, alters reading frame.
Nucleic Acid Biology IIDNA replication, key enzymes:1) Helicase – unwinds double helix @ origin (A-T rich)2) Primase – establishes RNA primer (polymerase requires free 3’ end)3) Topoisomerase – relieves supercoiling ahead of replication fork4) DNA Polymerase III – elongates growing chain in 5’3’ direction, *includes 3’5’
proofreading for each new nucleotide5) DNA Polymerase I – degrades RNA primers (5’3’ exonuclease) and replaces with
DNA 6) Ligase – seals nicks in DNA between completed fragments7) Telomerase – RNA-dependent DNA polymerase, extends chromosome ends
Nucleic Acid Biology III
Regulation of gene expression (DNA access):• Promoter – directly upstream from gene
• TATA box, mutations alter transcript levels
• Enhancers/Silencers – location varies dramatically due to DNA looping (may be VERY far, or even WITHIN gene)• Positive and negative transcription regulators will
bind here
• Chromatin architecture – histones (+) package DNA and actively regulate access of relevant enzymes• Heterochromatin – condensed, inactive• Euchromatin – open, active
DNA Repair (and clinical correlates):• Nucleotide Excision Repair – thymidine dimers, UV exposure xeroderma pigmentosum
• Base Excision Repair – cytosine deamination
• Mismatch Repair – newly synthesized, **unmethylated strand is proofed for mismatched base pairs hereditary nonpolyposis colon cancer
Nucleic Acid Biology IV
RNA processing in eukaryotes:
• hnRNA mRNA (occurs in nucleus)
• 7-methylguanosine cap @ 5’ end
• 3’ poly-AA tail (AAUAAA via poly-A polymerase)
• Splicing - intron removal (spliceosome, snRNPs, lariat structure)
5’ AAAAAAAA
1) Protection from cytosolic anti-viral exonucleases
2) Regulation of mRNA longevity (eg. protein lifespan)
5’ AAAAAAAA
• 20,000 – 40,000 genes >150,000 proteins, how?• Alternative splicing, post-translational processing
Energy Metabolism ICytoplasm
Glucose
Glucose-6-P
Mitochondrial Matrix
Acetyl-CoA
TCA
2x Pyruvate
Glycolysis
Fermentation
2NAD+, 2ADP, 2Pi 2NADH, 2ATP
3NADH, 1FADH2, 2CO2, 1GTP
e- TransportATPFatty Acids
β Oxidation
Ribulose-5-P Pentose Phosphate (HMP) Shunt
NADPH, Nulceotide sugars
Lactate Anaerobic
Aerobic
Fatty Acid Synthesis
Gluconeogenesis
NET:• Anaerobic glycolysis =
2ATP/ glucose
• Aerobic Oxidative Phosphorylation = 34-36ATP/ glucose
PyruvateDehydrogenase Complex I
Pyruvate Acetyl-CoA
NAD+, CoA
NADH, CO2
Key cofactors:1) Pyrophosphate (B1, thiamine, TPP)2) FAD (B2, riboflavin)3) NAD (B3, niacin)4) CoA (B5, pantothenate)5) Lipoic Acid
*** Contrast thiamine function with biotin Decarboxylation vs. carboxylation
PDH deficiency• Congenital or acquired
• **think alcoholic B1 deficiency
• Lactic acidosis neurological defects
• Tx: ↑ ketogenic nutrients (Lysine, Leucine) ↑ dietary fat content
NET: bypass pyruvate, enter TCA directly
- PDH- α ketoglutarate
dehydrogenase- branched chain ketoacid
dehydrogenase
- Pyruvate carboxylase- Acetyl-CoA carboxylase
Pyruvate Acetyl-CoA
NAD+, CoA
NADH, CO2
PyruvateDehydrogenase Complex II
Thiamine (B1) deficiency (Alcoholics) = Wernicke-Korsakoff Syndrome
A) WernickeLesion = foci of
hemorrhage/necrosis @ mamillary bodies + periaqueductal grey matter
1) Ophthalmoplegia (III, IV, VI)2) Ataxia3) Confusion
** Reverse after thiamine admin!
A) KorsakoffLesion = deterioration @ dorsal medial
nucleus (thalamus)
1) Memory loss, anterograde amnesia2) Confabulation – fabricated stories to fill
memory gaps, pts actually believe these events!
** Typically permanent!
Molecular biology: impaired glucose utilization via defective decarboxylation rxns.. PDH, α ketoglutarate DH, transketolase
Energy Metabolism ICytoplasm
Glucose
Glucose-6-P
Mitochondrial Matrix
Acetyl-CoA
TCA
2x Pyruvate
Glycolysis
Fermentation
2NAD+, 2ADP, 2Pi 2NADH, 2ATP
3NADH, 1FADH2, 2CO2, 1GTP
e- TransportATPFatty Acids
β Oxidation
Ribulose-5-P Pentose Phosphate (HMP) Shunt
NADPH, Nulceotide sugars
Lactate Anaerobic
Aerobic
Fatty Acid Synthesis
Gluconeogenesis
NET:• Anaerobic glycolysis =
2ATP/ glucose
• Aerobic Oxidative Phosphorylation = 34-36ATP/ glucose
Electron Transport Chain1 NADH 3 ATP, 1 FADH2 2 ATP
I
II
III IV
Mitochondrial matrix
Intermembrane Space
NADH NAD+
FADH2 FADH
e-
ATP Synthase
O2H2O
Q C
H+
H+ H+
ADP, Pi ATP
H+H+
H+
H+
H+
H+
O2
Electron Transport Chain1 NADH 3 ATP, 1 FADH2 2 ATP
I III IV
Mitochondrial matrix
Intermembrane Space
NADH NAD+e-
ATP SynthaseQ C
ADP, Pi ATP
H+H+
H+
H+
H+
H+
CN-
Oligomycin
DNP
1) Cyanide (CN-): e- transport inhibitor2) Oligomycin: ATP synthase inhibitor3) Dinitrophenol (DNP): Uncoupling agent
Cell Cycle + Division
Key concepts:
G1 S checkpoint via Cyclins, CDKs
Tumor suppressors:
Rb - phosphorylated by Cyclin D:CDK4
p53 - most common neoplastic mutation1) Stop cell cycle2) Promote DNA repair3) Promote apoptosis
Genetics
Hardy-Weinberg Equilibrium
1) No mutation
2) No natural selection
3) Random mating
4) No migration
For a gene with 2 alleles: (A, a)
p + q = 1
p2 + 2pq + q2 = 1
Ex: white irises are a recessive trait determined by a gene with two alleles (A, a). If 16 people out of 100 have white irises, what is the frequency of the carrier state?
aa = 16/100 = 0.16 = q2
q = 0.4, p = 0.6Aa = 2pq = 2 x 0.4 x 0.6 = 0.48
Genetics, Pedigree AnalysisWhat type of inheritance?
Identify carriers
Could this be autosomal dominant?
Incomplete Penetrance
Key questions:
1) Does the disease skip generations?
2) Does it appear equally in males and females?(transmission patterns)
Genetics, Pedigree Analysis
- Disease appears in every generation
- No male-to-male transmission
- 100% male-to-female transmission
- For females with disease, 50% of children are affected
- X linked dominant
1) Many anti-retroviral drugs combat nascent HIV infection by targeting the critical enzyme necessary for viral replication. Which of the following endogenous enzymes might be expected to experience some degree of cross-reactivity to these therapies?
A) DNA Topoisomerase
B) RNA Polymerase
C) Reverse Transcriptase
D) Telomerase
2) Which of the following accurately describes synthesis of the most abundant type of RNA in the cell?
A) Synthesized by DNA Pol III in the cytoplasm
B) Synthesized by RNA Pol II in the nucleus
C) Synthesized by RNA Pol I in the nucleolus
D) Synthesized by RNA Pol III in the golgi
3) Which of the following is true regarding the relationship between Prader-Willi and Angelman’s syndrome?
A) They are due to a mutation on the X chromosome and represent mosaicism
B) They are two types of muscular dystrophy
C) They are due to deletion of differentially methylated alleles of the same gene
D) They are co-dominant
4) Which of the following effects will be noted when oligomycin is applied to cells undergoing exclusively anaerobic metabolism?
A) ATP output will increase
B) There will be no change in ATP production
C) Electron transport will eventually cease, and a large proton gradient will build up across the inner mitochondrial membrane
D) Oxygen consumption will increase
5) Which of the following cellular conditions activate the pyruvate dehydrogenase complex?
A) High ATP
B) Elevated Acetyl-CoA
C) Increased NAD+/NADH ratio
D) Decreased Ca2+
E) Reduced Alanine concentration
6) A young infant is found to have prominent epicanthal folds, a simian crease, and a flat facial profile. An abdominal xray demonstrates air on either side of the pyrloric region. This disorder is likely caused by which of the following?
A) Maternal infection with a parasite found in cat feces
B) An enzyme deficiency
C) A trinucleotide repeat expansion
D) A meiotic non-disjunction event
7) Hexokinase and glucokinase both catalyze the same reaction (generation of glucose-6-phosphate from glucose). Which of the following best describes the difference between these enzymes?
A) Glucokinase has a ubiquitous distribution, whereas hexokinase occurs only in the liver
B) Hexokinase has relatively high affinity, whereas glucokinase has low affinity
C) Hexokinase is induced by insulin
D) Glucokinase has a relatively low capacity, while hexokinase has a high capacity
8) Fermentation occurs under anaerobic conditions to serve which of the following purposes?
A) To generate ATP directly in the absence of oxygen
B) To generate NADH for use in the electron transport chain
C) To regenerate NAD+ so that glycolysis may continue in the absence of oxygen
D) To produce Acetyl-CoA for use in the mitochondria
9) A homeless patient appears in the ER visibly intoxicated with alcohol on his breath. He is disoriented as to time and place, on physical exam his ocular cranial nerves do not function normally and he demonstrates an ataxic gate. Which of the following processes is impaired?
A) Electron transport chain
B) α ketoglutarate decarboxylation
C) Lactate formation
D) Pyruvate decarboxylation
10) A young child presents with coarse facial features, clouded corneas, and restricted joint movement. Laboratory exams demonstrate a high plasma level of lysosomal enzymes. Which of the following describes the underlying biochemical defect in this disease?
A) Impaired glycolysis in skeletal muscle cells
B) Defective mannose-6-phosphate addition at the golgi apparatus
C) Defective microtubule polymerization in phagocytic cells
D) Impaired collagen synthesis in the extracellular compartment
11) A child presents with a history of retardation, aggressive behavior, self-mutilation, and symptomatic gout. Laboratory examination reveals hyperuricemia. What is the correct diagnosis?
A) Niemann-Pick disease
B) Adenosine deaminase deficiency
C) Lesch-Nyhan syndrome
D) Osteogenesis imperfecta
12) In Huntington’s disease, the severity of the symptomatology worsens and the age of onset becomes earlier with each successive generation. This is an example of what phenomenon?
A) Codominance
B) Anticipation
C) Pleiotropy
D) Loss of heterozygosity
13) A young child presents with a history of progressive neurodegeneration and developmental delay. Upon exam you notice a cherry-red spot on the macula. If this child comes from an Ashkenazi Jewish background, the most likely diagnosis is which of the following:
A)Pompe’s disease
B) Tay-Sachs disease
C) Fabry’s disease
D) Hurler’s syndrome
