Cell & Molecular Biology Outline

1) Cell Cycle
a. Interphase (Growth)
i. G0 – most cells in body, some permanently, while others go in and out
1. Absence of mitogen stimulation
2. Special non-dividing phase
3. Examples:
a. Neurons, skeletal muscle cells
b. Liver cells (divide if stimulated)
c. Fibroblasts, lymphocytes – enter and exit G0
d. Bone Marrow, GI epithelial cells, hair follicles – rarely enter G0 – Labile cells
i. Labile cells are most effected by chemotherapy
ii. G1 – First growth phase
1. Length varies
2. Synthesis of proteins, organelles
3. Mitogens stimulate G1
a. Extracellular signaling molecules that stimulate cell division and function via
cyclin dependent kinases (Cdks)
4. Growth Factor – stimulate growth size
iii. S – DNA Synthesis; chromosomes to two sister chromatids
iv. G2 – Second growth phase
b. M – Mitosis
i. Shortest portion
1. Prophase – Chromosomes condense and spindle fibers form
2. Prometaphase – chromosomes organize on mitotic spindle
3. Metaphase – chromosomes line up on metaphase plate
4. Anaphase – chromosomes separate to opposite ends of the cell
5. Telophase – spindle breaks down and cell begins to divide
c. Cell Cycle Control
i. Checkpoints – also called restriction points that won’t be passed by cell unless conditions are
appropriate
1. G1-S (prior to S phase entry) – cell commits to DNA synthesis and cell division
a. Cdk activity is suppressed during G1
b. Mitogens activate Cdk to drive entry into S phase
i. Interact with cell surface receptors
ii. Activate Intracellular pathways
iii. Increases G1 cyclin levels
iv. Increase Cdk activity
c. Cyclin-Cdk complexes activate E2F proteins
i. Transcription factors that bind DNA promoter regions and activate
genes for S phase
d. E2F is normally inhibited by retinoblastoma proteins (Rb)
i. G1-S-Cdk phosphorylation of Rb releases inhibition
1. Rb regulates cell growth → Tumor Suppressor
e. DNA damage can arrest cell division (prevent entrance to S phase) to allow for
repair and prevent development of mutant/cancer cells
i. DNA damage initiates signaling pathways – that effect whether or not a
cell proceeds
 f. Pathways for arresting cell cycle at G1-S due to DNA damage lead to protein
phosphorylation causing arrest in the cell cycle/growth of cell
i. ATM Pathway: activated by double strand breaks in DNA
1. ATM gene mutation
ii. ATR: single stranded breaks
g. P53 Protein is the major target of both ATM/ATR systems
i. Phosphorylated after DNA damage, which prevents p53 breakdown and
increases its levels and increases activity (p53 is normally unstable and
rapidly breaks down)
ii. P53 induces transcription of p21 → p21 binds to Cdks → inhibits Cdk
activity
1. Blocks cell progression through the cell cycle
iii. P53/p21 are tumor suppressors because prevent abnormal cell growth
2. G2-M (before mitosis)
3. M phase (prior to anaphase and cytokinesis)
ii. First Checkpoint: Late G1 (G1-S)
1. Where cell commits to cell cycle/growth
iii. Key elements:
1. Cyclin Dependent Kinases (Cdks)
a. Central component of cell cycle control
b. Kinase Enzymes (lead to phosphorylation of other proteins)
c. Always present in cells but inactive – wait to drive cell through the cycle
d. Depend on cyclins to activate
2. Cyclins – regulatory proteins that activate Cdks
a. Many classes/subtypes that vary during cell cycle
i. Cyclin D – Rises throughout cell cycle
ii. Cyclin E – Highest at G1-S transition
iii. Cyclin A – Highest during G2
iv. Cyclin B – Rises at G2-M transition
3. Cyclin-Cdk complexes phosphorylate regulatory proteins to allow progression through
the cell cycle
d. Cell Cycle Clinical Correlates
i. Retinoblastoma – rare childhood eye malignancy due to mutation in RB1 gene which codes for
the Rb protein
1. Abnromal Rb → leads to unregulated cell growth via E2F
ii. Li-Fraumeni – multiple malignancies at early age
1. Sarcoma, Breast, Leukemia, Adrenal Gland
2. Mutation in tumor suppressor gene TP53 that codes for p53 protein
3. Result that cell cycle is NOT arrested to allow for DNA repair, leading to an accumulation
of damage and malignancy

Genes to Proteins

2) DNA Replication
a. DNA Overview
i. contains genetic code, located in nucleus of eukaryotic cells and cytoplasm of prokaryotic cells;
replicated for cell division and growth
ii. Structure – Double stranded, with Sugar backbone, connected by phosphate and Nitrogenous
base
iii. Base Pairing – antiparallel 5’ and 3’ ends
 1. DNA – AT (2H Bonds) and GC (3H Bonds)
2. RNA – AU and GC
iv. Nucleotides
1. Synthesized as monophosphates → Converted to triphosphate → added to DNA
b. Replication - big picture: two strands separate and two new strands will be synthesized
i. Has directionality – all of the energy comes from breaking the triphosphate bond – phosphate
bond is at 3’ end, so DNA polymerase will always add to the 3’ end
1. Always occur in 5’ to 3’ direction (nucleotides always added to the 3’ end)
ii. Semi-conservative – one old and one new strand
iii. Origin of Replication
1. Specific DNA sequences that attract initiator proteins and are easy to unwind/open
a. AT rich sequences have fewer bonds and are easier to open
2. Enzymes
a. DNA Helicase: unwinds DNA double helix, hydrolyzes ATP
b. ssBP assist helicase by stabilizing and straightening single strands
iv. Replication Fork:
1. Leading Strand – continuous addition of nucleotides to the 3’ end
2. Lagging Strand – Okazaki fragments with multiple primers and DNA ligase, discontinuous
v. DNA Polymerase
1. Bacteria
a. DNA Pol I-IV
i. Pol III: synthesizes new strands
ii. Pol I: Removes RNA primers
2. Eukaryotes
a. DNA polymerase α, β, γ, δ, ε
b. Pol γ: located in the mitochondria (have own DNA)
vi. DNA Primers:
1. Short nucleotide sequences that enable DNA polymerase to initiate replication
a. Formed at point of initiation of anew chain
2. DNA Primase: makes primers
3. Primers contain RNA with U instead of T; these are eventually removed and replaced
with DNA
4. RNA Primers are removed and replaced with DNA
a. Prokaryotes: DNA pol I
b. Eukaryotes DNA pol δ
vii. DNA Ligase: creates phosphodiester bonds to join Okazaki fragments
viii. Topoisomerase: prevents DNA tangling
1. Breaks DNA and reseals to relive tension/twists
2. Topo I – breaks single strands and reseals
3. Topo II – breaks double stands and reseals
ix. Topoisomerase Clinical Correlates
1. Quinolone antibiotics – inhibits prokaryotic topoisomerases
2. Chemotherapy Agents – target Eukaryotic topo
a. Etopside/Teniposide
b. Irinotecan, topotecan
c. Anthracyclines
x. Telomerase – extends the 3’ end of DNA; to allow DNA polymerase to complete lagging strand
and avoid the loss of genetic material
 1. Telomeres are nucleotide sequences at the end of chromosomes that contain TTAGGG
sequences
2. There is no place for RNA primer on lagging stranding
3. Telomerase recognizes telomere sequences and adds them to new DNA strands
4. RNA template that is uses to synthesize telomere DNA – RNA-dependent DNA
polymerase; similar to reverse transcriptase
5. Telomerase is found in hematopoietic stem cells – to allow controlled indefinite
replication as well as other cells that divide indefinitely (epidermis, hair follicles,
intestinal mucosa)
6. Telomerase allows immortality in many cancers
c. Proofreading – DNA polymerase can correct errors
i. DNA polymerase adds the wrong nucleotide, can move backwards in the 3’ to 5’ direction to
correct error:
1. Exonuclease: remove the incorrect nucleotide
a. DNA polymerase has 3’ to 5’ exonuclease activity, significantly reducing the
error rate
3) DNA Mutations – errors in DNA, simple (one of few base) or complex (gene deletions or translocations)
a. Affected Cells
i. Germ Line – DNA of Sperm/eggs – transmitted to offspring and found in every cell in the body
ii. Somatic – acquired during the lifespan of cell – NOT transmitted to offspring
b. Types
i. Point Mutations – single base change
1. Transition (more common)
a. Purine to Purine (A to G)
b. Pyrimidine to pyrimidine (C to T)
c. Wobble effect allows altered bases in 3rd position to code for same amino acid;
allows some transitions to
2. Transversion
a. Purine to pyrimidine, vice-versa
3. Classifications:
a. Silent – nucleotide substitution code for same amino acid; often base change in
3rd position that does not cause a change in protein
b. Nonsense – nucleotide substitution that results in early stop codon (UGA, UAA,
UAG) that leads to a truncated protein
c. Missense – substitution leads to a different amino acid in the final protein – that
may change the function of the protein
i. Sickle Cell Anemia – root cause is missense mutation in β-globin gene
that is single base substitution → changes adenine to thymine →
substitutes valine for glutamate; Glu → Val – the change in shape cause
RBCs to sickle
ii. Insertions/Deletions – addition or subtraction of nucleotides that alters the size of DNA leading
to an altered protein product (e.g. Cystic fibrosis – delta F508 – deletion of 3 DNA bases → loss
of phenyalanine → abnormal protein folding)
1. Frameshift mutation: insertion or deletion that alters the reading frame and causes a
dramatic change in the product – basically insertion or deletion in # of nucleotides not
divisible by 3
a. Misreading of downstream nucleotides and codons, significant changes in
protein:
i. Many amino acids change downstream of the mutation
 ii. Early stop codon → truncated protein
iii. Loss of strop codon → elongated protein
b. Clinical Correlates:
i. Tay Sachs
1. Frameshift in gene for hexosaminidase A
ii. Duchenne muscular dystrophy
1. Dystrophin gene mutation causes absence of functional
dystrophin
c. Additional Types:
i. Large Segmental Deletion (unequal crossover in meiosis) – leads to loss of function where a
gene is short than normal or missing (α-thallesemia)
ii. Splice Site (5’ or 3’) – Variable effects ranging from new addition or deletion of a few amino
acids to deletion of an entire exon
1. Β-thallesemia; gaucher’s, tay-sachs, etc.
d. DNA Slippage – Slipped-Strand Mispairing occurs in areas of repeated nucleotide sequences that are
close together because of inadequate mismatch repair → can lead to insertions/deletions → frameshift
mutations
i. Wrong repeated sequences bind together pushing nucleotides out during replication this can
result in two different length strands of DNA
ii. Trinucletotide Repeat Expansion Disorders lead to extra repeats in a gene – as homologous
recombination occurs – as these are inherited down generation, diseases are more severe
1. Fragile X syndrome
2. Friedreich’s Ataxia
3. Huntington’s Disease ((CAG)n) – 3-5th decade of life
4. Myotonic Dystrophy
iii. Microsatellite instability
1. Microsatellite are short segments of DNA
2. Mismatch repair enzyme failure cause instability (variation) in size of the segments
among cells
a. Leads to colon cancer

4) DNA Repair – cells contain numerous repair enzymes and mechanisms

a. DNA Damage – occurs in life of cell due to heat, UV radiation, chemicals, free radicals; however rarely
leads to permanent damage
i. Distinct from Mutations in the genetic material
ii. Types:
1. Depurination – loss of purine base (free adenine or guanine)
a. Occurs spontaneously
2. Deamination - loss of amine groups (cytosine)
a. Occurs spontaneously
b. Cytidine loses the amine group, creating uracil
3. Free radicals or radiation damage bases rings
a. Oxidative damage, methylation, hydrolysis
b. DNA Repair mechanisms
i. Single Strand
1. Base Excision Repair – recognize specific base errors from deamination, oxidation, or
open rings; has numerous variations, and functions throughout the cell cycle
a. Mechanism:
 i. DNA Glycosylases (several enzymes) that remove damaged base, create
a baseless, “apurinic” or “apyrimidic” nucleotide
1. Glycosylase breaks the glycosidic bond – allowing the base to go
free
ii. AP endonuclease – recognizes nucleotides without a base (closer to 5’
end)
1. Attacks 5’ phosphate end of DNA strand, nicks damaged DNA
upstream, creating a 3’ OH end adjacent to the site
iii. AP Lyase – attack 3’ hydroxyl end of ribose sugar (closer to 3’ end)
iv. DNA Polymerase – adds new nucleotides (complementary), adds to
3’OH ends
v. DNA Ligase seals strands
2. Nucleotide Excision Repair removes bulky DNA damage (multiple bases)
a. Pyrimidine dimers commonly caused by UV radiation
i. Thymidine gets linked together into cyclobutene pyrimidine dimers;
b. Active in the G1 phase prior to DNA synthesis
c. Endonucleases remove multiple nucleotides
d. Clinical Correlate: Xeroderma Pigmentosum
i. Occurs due to defective nucleotide excision repair and leads to a VERY
high risk of skin cancer (may develop in childhood)
ii. Presents in infancy or childhood as extreme sensitivity to UV rays with
easy sun-burning, freckling, dry skin, and changes in pigmentation
3. Mismatch Repair – identifies incorrectly placed base/nucleotides (when proofreading
misses errors like insertions, deletions, incorrect matches); there is no damage to the
base, so it is not recognized by repair systems; Backup to proofreading
a. Occurs after DNA synthesis during the S and G2 phase - newly synthesized
strand are compared to template strand, with nucleotide errors removed and
resealed
b. Maintains microsatellite stability – “Microsatellites” are repeating segments of n
nucleotide segments
i. DNA slippage may occur at repeats resulting in a mismatch; MMR
c. Microsatellite instability results if MMR system are deficient (results in different
numbers of microsatellites)
i. Clinical Correlates: Lynch Syndrome (Hereditary Non-Polyposis
Colorectal Cancer)
1. Due to germline mutation of DNA mismatch repair enzymes
(90% due to MLH1 and MSH2 mutations) that leads to colon
cancer via microsatellite instability with an 80% lifetime risk
2. Hallmark is cancer cells with microsatellite instability
ii. Double Strand – often from exogenous sources (radiation therapy, ionizing radiation)
1. Homologous End Joining – uses sister chromosome as a template
2. Non-Homologous End Joining – use many proteins to re-join broken ends
a. DNA pol γ and μ re-extend the ends, with many other enzymes
b. No template is used, so this is highly error-prone
c. Clinical Correlates:
i. Fanconi Anemia – inherited aplastic anemia with >13 genetic
abnormalities
1. Involve DNA repair enzymes
a. Hypersensitivity to DNA damage
 b. Cells Vulnerable to cross linking
ii. Ataxia Telangiectasia – defective NHEJ
1. Mutation in ATM gene on chromosome 11
a. Ataxia Telangiectasia Mutated gene that is responsible
for repairing double stranded DNA breaks via NHEJ
2. Results in DNA that is hypersensitive to ionizing radiation with
accumulation of DNA damage in the CNS, skin, and the immune
system
3. Presentation: healthy through first year and begin walking at a
normal age, with slow development
a. Result in progressive motor coordination problems,
most patients in wheelchair by 10 years old
b. Recurrent sinus and respiratory infection as well as
Telangiectasias (skin problems)
c. HIGH RISK OF CANCER because DNA damage occurs in
cells that are exposed to ionizing radiation

5) Transcription

Overview Tables:

Prokaryotes
Gene Regions - May be polycistronic
- Genes are continuous coding
regions
- Very little spacer (noncoding)
DNA between genes
RNA Polymerase 1 Core α2ββ’ enzyme
Initiation of transcription - Promoter (-10) TATAAT and (-
35) Sequences
- Sigma (σ) initiation subunit
required to recognize
promoter
Eukaryotes
- Always monocistronic
- Genes have exons & Introns
- Larger spacer non-coding
regions between gene
RNA Pol I: rRNA
RNA Pol II: mRNA, snRNA
RNA Pol III: tRNA, 5S RNA
- Promoter (-25) TATA and (-
70) CAAT
- Transcription factors TFIID
bind to the promoter

mRNA synthesis Template read 3’ to 5’; mRNA synthesized 5’ to 3’; gene sequence is specified
from the coding strand 5’ to 3’; transcription begins at a +1 base
Termination of transcription Stem and Loop + UUUUU Not well characterized
Stem and loop + rho factor
Relationship of RNA transcript to RNA is antiparallel and complementary to DNA template strand; RNA is
DNA identical (except U substitutes for T) to DNA coding strand
Post-transcriptional processing of NONE In Nucleus:
hnRNA 5’ cap (7-MeG)
3’ tail (poly-A sequence)
Removal of introns (alternative
splicing yields variants of protein
product
Ribosomes 70S: (30s and 50S) made of rRNA and 80s: (40S and 60S) rRNA and protein
protein
tRNA Cloverleaf secondary structure
- Acceptor arm (CCA) carries the amino acid
 - Anticodon arm; anticodon complementary and antiparallel to codon
in mRNA

a. Overview:
i. Not all DNA is transcribed – most DNA is not turned into protein
ii. RNA polymerase initially finds and binds to the promoter (not the coding region)
iii. “Gene” = dsDNA but only one strand is used as template for transcription
1. Template strand is used for transcription is complementary and antiparallel (read 3’→5’)
2. Coding stand is not used, but is identical to mRNA (T/U) – this is where you see the TATA
and CAAT box ; sequence reported in databases; assume that Left is 5’ and Right is 3’
iv. DNA read 3’ → 5’; RNA synthesize 5’→3’
v. Transcription proceeds from PROMOTER to TERMINATOR
vi. RNA Types: mRNA, rRNA (most abundant), tRNA, snRNA
vii. Promoter regions are NOT transcribed (transcription begins at the +1 site)
b. RNA Polymerases (Prokaryotic vs. Eukaryotic)

Prokaryotes Eukaryotes
Single RNA polymerase: α2ββ’ 3 RNA Polymerase:
- RNA Pol 1: rRNA (Nucleoulus – makes
ribosomal subunits)
- RNA Pol 2: mRNA/hnRNA, some snRNA
- RNA Pol 3: tRNA, 5S rRNA
Sigma factor required to initiate at promoter No sigma, TF (TFIID) bind before RNA polymerase
May require ρ to terminate NO ρ required
Inhibited by rifampin (TB & Meningitis - prophylaxis) RNA Pol 2 (mRNA) inhibited by α-aminitin
& Actinomycin D (mushrooms) & Actinomycin D

c. Transcription Mechanism/Apparatus
i. Promoter: precedes gene, landing strip for RNA Pol
1. Upstream vs. Downstream: relative to coding strand
a. Upstream: Towards 5’ end of coding strand
b. Downstream: towards 3’ end of coding strand
d. Types of RNA
i. mRNA
1. Prokaryotic Transcription Unit
a. Key Facts:
i. Polycistronic – multiple genes in a single mRNA
1. Each gene preceded by 5’UTR with Shine-Delgarno sequence, 5’
start codon, and 3’ stop codon
2. In polycistronic gene, ribosomes translate each gene
independently
ii. No Introns
iii. Transcription and Translation can occur simultaneously (Ribosome reads
5’ to 3’ and synthesizes from amino to carboxy terminal)
iv. Shine-Delgarno Sequence – a sequence in the 5’ UTR that binds to
prokaryotic ribosomal RNA
v. 3’ Stem-loop structures involved in transcription termination
b. Features of Prokaryotic mRNA
i. 5’-UTR and 3’-UTR – contain no information that is translated into
protein
 ii. Promoter at -35 sequence region, -10 TATA box region
iii. Coding Region – region between 5’UTR and 3’UTR
2. Eukaryotic Transcription Unit
a. Key Facts:
i. Monocistronic
ii. Maturation
1. Caping
2. Poly-adenylation
3. Splicing
iii. Transcription in nucleus; translation in the cytoplasm
b. Genes more widely spaced and have their own promoter
c. Information is fragments
i. Exon = expressed and contain protein encoding information
ii. Intron = intervening
d. Promoter region
i. CAAT Box (-70) on coding strand
ii. TATA Box (-25) on coding strand
e. Pre-mRNA contains intron sequences (that will be removed before it is mature)
f. Maturation: Pre-MRNA is hnRNA
i. Capping: 7-methyl-guanosine Cap provides protection to the mRNA and
identifies the species as an mRNA
1. Co-transcriptional
ii. Poly A Addition – protects mRNA and helps mRNA exit the nucleus
1. Post-transcriptional
iii. Splicing by Spliceosome (snRNA) happens in the nucleus
1. Spliced out intron forms Lariat-loop which is degraded in the
nucleus
2. Both post, and co transcriptional
3. snRNA (Small Nuclear) and snRNP make up the spliceosome
3. Alternative Splicing (Specific to Eukaryotes) of mRNA
a. One single mRNA, with one start and one stop codon and multiple splicing
variations that bring together different exons
i. One hnRNA can make many different proteins:
1. Membrane vs. secreted Ig
2. Tropomyosin variants in muscle
3. Dopamine receptors in the brain
ii. rRNA
1. Prokaryotic vs. Eukaryotic Ribosomes (rRNA)
a. Prokaryotic (50S + 30S = 70S)
i. 16S RNA of 30S subunit = recognizes the shine-delgarno sequence to
initiate translation
ii. Different Antibiotics block different subunits of the prokaryotic
ribosome
b. Eukaryotic (60S + 40S = 80S)
i. rRNA is made by RNA Pol 1 (except 5s RNA = RNA Pol III)
ii. 40S subunit recognized by the 7-methyl Cap
iii. tRNA – transfer RNA with cloverleaf-like structure functions to pick up amino acids and
recognize the codon on the mRNA
1. 5’ Phosphate
 2. 3’ OH – CCA residue that covalently binds to the Amino Acid
a. Aminoacyl tRNA Synthtase attach activated amino acid to the 3’-OH
3. Anti-codon – complementary and antiparallel to codon on mRNA
4. tRNA contains modified base –
a. there are T’s in tRNA

6) The Genetic Code and Translation

a. The Genetic Code – which bases code a particular amino acid
i. Universal
ii. Degenerate: UUU & UUC = Phenylalanine
iii. Unambiguous: UUU = Phe always
iv. Start Codon: AUG (RNA), ATG (DNA)
v. Stop Codons: UAG, UGA, UAA
vi. Wobble Hypothesis – third base is less likely to cause a mutation that is detrimental to a protein
vii. CAG repeats are seen in Huntington’s Disease
b. Translation (in the cytoplasm)
i. tRNA charging (3’ end, and anticodon sequence are key)
1. Aminoacyl-tRNA Synthetase takes amino acid and attaches it to tRNA at 3’-OH
a. Anti-codon on Aminoacyl tRNA specifies which amino acid; there are several
aminoacyl-tRNA synthetases in the cell
b. Codon Translation – anticodon portion binds complimentary and antiparallel
ii. Protein Synthesis
1. Peptide Bond Formation – amino group bound to the carboxyl group through
dehydration reaction – dictates the primary structure
a. Peptide bond formation between carboxyl group of amino acid in P site and
amino ground of the AA in the A site; synthesis occurs from the amino to the
carboxyl terminus
2. Steps of translation
a. Initiation, Elongation, and termination each of which require specific factors –
initiation factors (IF), elongation factors (EF), termination factors (release
factors), as well as GTP
i. Initiation:
1. Small ribosomal subunit binding to mRNA
a. Prokaryotes: 16S rRNA subunit binds to Shine-Delgarno
sequence in 5’UTR of mRNA
b. Eukaryotes: small subunit binds to the 5’ cap structure
and slides to AUG start codon
2. 1st chaged tRNA (AUG – methionine) becomes bound to AUG
start codon by base pairing with its anticodon – in both
prokaryotes and Eukaryotes, initial binding occurs between the
charged-tRNA and the P (peptidyl) site of the ribosome
a. Prokaryotes: fmet
b. Eukaryotes: MET
3. Large subunit binds, completing the initiation complex
4. Key ribosomal binding sites:
a. P site – Peptidyl Site – binding site for growing peptide
chain
 b. A site – aminoacyl site – each new incoming tRNA binds
carrying activated amino acid
ii. Elongation – 3 step cycle repeated for each amino acid, uses 4 high
energy bonds (2 from ATP and 2 from GTP); ribosome moves in the 5’ to
3’ direction, protein is synthesized from AMINO to CARBOXYL terminus
1. Step 1: charged tRNA binds in A site – determined by mRNA
codon in the A site
2. Step 2: Peptidyl transferase forms peptide bond between AA in
P and A site; breaks bond between tRNA in P-Site and growing
peptide, leaves attached to tRNA in A site
3. Step 3: Ribosome translocates 3 nucleotides along the message
putting the growing peptide back in the P site
a. Translocation requires elongation factor-2 (eEF-2)
which can be inactivated in eukaryotes by pseudomonas
and diphteria toxins
b. Shiga/Shiga-like toxin stop protein synthesis in
eukaryotes
iii. Termination – Stop codon moves into the A site, peptidyl transferase +
release factor hydrolyzes completed protein at the P site, complex
dissociates and is reused in future protein synthesis

7) Cell Membranes
a. Phospholipid Bilayer is fundamental structure of the plasma membrane
i. Phospholipid – 2 hydrophobic fatty acid chains linked to phosphate containing hydrophilic head
group
1. Amphipathic molecules that spontaneously form micelles and liposomes
2. Membranes contain 5 major phospholipids (clasifed by the type of fatty acid and polar
head group; in total account for <50% of the lipid in membranes
3. Assymterically distributed between two halves of lipid bilayer
4. FA chains often unsaturated and have kinks that increase fluidity
5. Cytosolic face has a net (-) charge
6. Erythrocytes provide the first proof that biological membranes consisted of lipid bilayers
ii. Steroids (i.e. Cholesterol) – rigid ring structure found within the membrane with head group
located on surface
1. Cholesterol buffers membrane fluidity (↓ at high temp, maintains at low temp)
2. Portion of Lipid Rafts (dynamic structures that limit lateral diffusion and serve as
platform for signal transduction)
a. Cholesterol, glycolipids, sphingomyelin
b. Recruit proteins (GPI-anchored proteins) and other molecules
i. Caveolae – subclass of lipid rafts associated with caveolin (peripheral
protein), function in endocytosis
iii. Glycolipids – ONLY in outer leaflet – carbohydrate portion exposed on outer surface
1. Glycocalyx – exposed carbohydrate portion protects cell surface from ionic/mechanical
stress, forms a barrier to invading microorganisms, participates in cell-cell interactions
iv. Proteins – function as receptors, membrane connections, intercellular connections, for
transmembrane transport, and cellular respiration
1. Integral Membrane Proteins – within the bilayer, often transmembrane (glycoproteins)
a. Exposed on both sides of membrane; integral alpha-helical portions (proins are
beta barrel channels in mitochondira)
 b. Covalently bound by lipids to stay in place
c. Inserted into ER during synthesis and transported to golgi in vesicles where
carbohydrate groups are added to the proteins
i. Glycophorin – heavily glycosylated transmembrane protein with N
terminal on outside of membrane
ii. Band 3 – anion transporter that spands membrane 14 times (N & C
terminals external to membrane)
d. Glycosylation important for cell-cell communication
2. Peripheral Membrane proteins – associate with membrane through protein-protein
interactions; can be important components that hep determine cell shape (SPECTRIN)
a. E.g. Actin, Ankyrin (links membrane and cytoskeleton), Band 4.1
b. Released from membrane by treatment with reagents
c. Glycosylphosphatidylinositol (GPI) Anchors – inserted into ER by bound C-
terminal transmembrane region and are often found in lipid rafts
d. Outer leaflet proteins synthesized/modified in secretory pathway in the ER;
inner leaflet protein synthesized on free cytosolic ribosome
3. Membrane protein mobility
a. Proteins + lipids move laterally; can NOT flip between inner and outer leaflets
b. Proteins immobilized by connection with cytoskeleton, other membrane
proteins, surface proteins on other cells, connections with EC matric
c. Mobility restricted for various reasons:
i. Specialized tissue function may require immobilization – i.e. microvilli
for nutrient absorption
ii. Cell junctions
iii. Lipid rafts stabilized by interactions with membrane proteins

8) Transmembrane Transport – various mechanisms require energy, others do not

a. Passive Diffusion – nonselective movement of a molecule across bilayer with NO energy
i. Molecules flow DOWN [gradient]
ii. Small, hydrophobic molecules: O2, CO2, Hydrophobic steroid hormones, small polar uncharged
molecules
b. Facilitated Diffusion – molecular flow down [gradient] with proteins that limit molecules contact with
inner hydrophobic region of membrane – polar charged molecules can cross membrane
i. Channel Proteins – form pores that allow diffusion of certain molecules
1. Porin – passage of ions/small polar molecules through mitochondrial membrane (i.e.
Aquaporin)
2. Ion Channel – role in transmission of electric impulses (i.e. NA+/K+ channel opening)
a. Rapid, selective transit, opening can be regulated
i. Ligand-gated ion channels (bound by neurotransmitters)
ii. Voltage-gated Channels (change in electric potential causes opening)
iii. Na/K Channel
ii. Carrier Protein – bind molecules to be transported, change conformation to allow molecular
passage
1. Sugar transport – GLUT has 12 membrane alpha helices; GLUT 4 incolved in diabetes
c. Active Transport – couples rxns (hydrolysis of ATP) which drive uphill transport of molecules in
energetically unfavorable directions
i. Ion Pumps (Na+/K+ ATPase, Ca2+ transport) – use energy from ATP to maintain ion gradients
1. ATP binding/hydrolysis drives conformational change in pumps
 a. Na+/K+ ATPase plays role in maintaining resting potential in cell membrane and
helps propagate electric signals;
i. Roles: establish gradient for action potential, Drive active transport,
maintain osmotic balance and cell volume
b. Ca2+ transport vital to cell signaling/muscle contraction
c. H+ pumps help create acidity of gastric fluids

9) Endocytosis – process by which cells take up macromolecules and particles from surrounding medium; includes
phagocytosis and pinocytosis
a. Phagocytosis – cell engulfs large particles (bacteria, debris, intact cells); defense against micro-
organisms, eliminate old/damaged cells – primarily neutrophils and macrophages
i. Actin based movement to extend pseudopodia to surround particle → membrane fuses to form
phagosome
ii. Phagosome-lysosome fusion to digest material with lysosmal acid + hydrolase
b. Receptor mediated endocytosis – selective mechanism to uptake macromolecules
i. EC material taken into transport vesicles to endosome + lysosome
1. Macromolecule binds receptor in, i.e. clathrin-coated pit
2. Internal signal binds adaptor proteins (i.e. clathrin)
3. Clathrin helps forms basketlike structure and invaginates, forming a clathrin coated
vesicle
a. Dynamin plays key role in vesicle budding; by assembling ring around neck of pit
4. Clathrin coated vesicle fuses with an early endosome for content sorting and transport
ii. Cholesterol uptake facilitated by receptor-mediated endocytosis
1. LDL transports cholesterol (has apo B100) and binds to receptor in clathrin coated pit,
transported to lysosome, broken down, and cholesterol released for use by the cell
2. Familial Hypercholesteremia occurs to to a failure to bind LDL to a receptor or a failure
to internalize LDL after binding
c. Clathrin-independent pathways
i. Caveolae carry out receptor mediated by acting as receptors – organized by Caveolin
ii. Macropinocytosis – endocytosis of lfuids by large vesicles
d. Fluid Phase Endocytosis – nonselective uptake of extracellular fluids and their contents
e. Protein Trafficking in Endocytosis – after internalization of clathrin-coated pits
i. Vesicles fuse with endosome mediated by Rab-GTP binding proteins and SNARE proteins
1. Early endosome sorts contents – some recycled to plasma membrane, others sent to
lysosome for degradation
a. Recycling – 50% of plasma membrane internalized every hour – need for quick
replacement, most of which is by recycling
2. Late Endosome – more acidic, fuses with lysosomal hydrolases from the Golgi, contains
contents sent for degradation; becomes lysosome after acquiring all lysosomal
components
ii. Receptor Down-Regulation – some receptors degraded, not recycled while others are recycled;
e.g. growth factor binding receptor is degraded, terminated the response
iii. Recycling in nerve impulses – action potential signals fusion of synaptic vesicles with plasma
membrane, release neurotransmitters to carry signal to postsynaptic cells – Empty clathrin-
coated vesicles, fuse with early endosomes to accumulate new supply of neurotransmitters and
are recycled to the plasma membrane
iv. Transcytosis – process of transferring an internalize receptor across cell to opposite side of
plasma membrane
10) Molecular Medicine
a. Cystic Fibrosis (cellular transport)
b. Mitochondrial Diseases (leber’s Hereditary Optin Neuropathy)
c. Gaucher’s Disease (Lysosome)

11) Organelles
a. Overview:
i. Organelles enable the cell to:
1. Specialize/Compartmentalize
2. Increase membrance surface area
3. Give cells characters (i.e. liver cell have a lot of ER and mitochondria to detoxify drugs)
4. Specialize and adapt to conditions (i.e. the amount of ER will adapt to the environment)
b. Endoplasmic Reticulum – ALL eukaryotic cells,
i. Structure: folded membrane, of sacks and tubules continuous with the nuclear membrane
ii. Function: synthesis of proteins and lipids
1. Rough Endoplasmic Reticulum – surface of ER covered with ribosomes that give it a
granular, rough appearance; site of protein synthesis
a. Ribosomes
i. Membrane bound ribosomes – on RER, produce proteins for secretion
from the cells, like protein hormones and digestive enzymes
ii. Free Ribosomes – in cytosol, mostly proteins used by the cell, for
instance those for metabolism and cell structure
b. Nissl Body – RER of neuron synthesizes neurotransmitters
c. Locations: cells that secrete proteins
i. Goblet cells, plasma cells, pancreatic beta cells
2. Smooth Endoplasmic Reticulum – portion without ribosomes performs lipid/steroid
synthesis, drug/toxin detoxification
a. Sarcoplasmic Reticulum is the SER in myocytes (stores calcium for muscle
contraction)
b. De Novo membrane sysnthesis happens here, where phospholipids and
transmembrane proteins are synthesized
i. New phospholipids are inserted into the cytosolic side of the SER,
cannot flip on their own
ii. Flipases facilitate flipping of phospholipids in the membrane to help
achieve equilibrium
iii. New phospholipids reach cell membrane in the form of vesicles that bud
off of the SER
c. Locations: Hepatocytes and steroid producing organs
i. Hepatocytes synthesize cholesterol, lipoproteins, detoxification
enzymes, and cytochrome P450 enzymes
ii. Steroid producing organs like the adrenal glands and gonads
d. Physiological assay’s are needed to understand how an individuals body
responds to toxins
c. Golgi Apparatus – Proteins are modified and sorted for transport to next destination
i. Proteins travel from ER in vesicles and are transported to the Golgi, empty contents
ii. Structure
1. Cis Golgi – closest to RER – vesicles enter here
2. Trans Golgi – closest to membrane, vesicles exit from this side
 iii. Function: proteins are sorted and shipped by the golgi through the addition of signal sequences;
modifications protext from degradation, direct to target, and allow for recognition by receptors
1. N-oligosaccharides on asparagine
a. Oligosaccharide attached to a nitrogen
b. N-linked Oligosaccharides are synthesized in ER and modified in the Golgi
(trimmed, sugars are added)
2. O-oligosaccharides on serine and threonine
a. O-linked – attached to oxygen
b. In the Golgi apparatus
c. i.e. Mucins
3. Mannose-6-phosphate on lysosomal proteins
a. Examples: acid hydrolases
b. Triggers packaging in trans-golgi
c. This is disrupted/abnormal in I-cell Disease
4. Inclusion cell disease: Autosomal recessive disorder
a. Signs/Symptoms: onset in first year of life, presents with growth failure
hypotonia, and motor delay
b. Findings: Mannose-6-phosphate is NOT found on lysosomal proteins, Acid
hydrolases are detected in blood/urine and are missing from lysosomes;
Lysosomes contain inclusions of undigested GAGs and glycolipids
c. Cause: Deficiency of N-acetylglucosaminyl-1-phosphotransferase
d. Endosomes – membrane bound compartments in cells formed by Endocytosis
i. Types of endocytosis
1. Receptor Mediated – cells take up molecules that bind receptors
2. Pinocytosis – cells take up extracellular liquid
3. Phagocytosis – cells extends pseudopods, encircles particles, key for immune defense
ii. Transport contents to lysosomes or back to cell membrane
e. Lysosomes:
i. Acidic
ii. Acid Hydrolase Enzymes (require acidic environment, breakdown substrates by hydrolysis)
1. Deficiency leads to lysosomal storage disease and a cellular build of macromolecules
iii. Role: Breakdown waste fast, carbs, proteins to generate simple compounds that can be used by
the cells
f. Peroxisomes – cellular organelles that contain oxidative enzymes and can generate hydrogen peroxide
and Catalase (oxidizes substances, detoxifies substance in liver cells)
i. Some Beta oxidation (mainly in mitochondria), preferentially long chain fatty acids
g. Proteasomes – destroy abberant (misfolded proteins)
i. Structure: Barrel shape of multiple subunity and require ATP
ii. Function: destroy ubiquitin marked proteins
iii. Clinical Correlate: may play role in Parkinson’s due to reduced ubiquitin-proteasome activity
leading to a toxic accumulation of proteins in neurons
12) The Secretory Pathway
a. Overview: series of steps for secretory proteins
b. Signal sequences – short molecular sequences on proteins undergoing synthesis, and are used to “pull”
free ribosomes to the ER membrane → creates RER
i. Proteins enter ER lumen, and will mostly be secreted, while some will go to the ER and other
organelles
ii. Short peptides on the N-terminal portion of protein and direct protein-ribosome to the
endoplasmic reticulum
 iii. Signal Recognition Particle (SRP) – ribonucleoproteins in the cytosol that complex with many
proteins and RNA, recognize signal sequences, and move proteins from the cytosol to the ER
1. SRP receptors exist on ER membrane and bind SRPs
2. Proteins are translocated through pore into the ER lumen
13) Coated Vesicles – protein coat on vesicle surface formed from specialized portions of membranes
a. Different coats used for different forms of traffic – like transport from cell surface or secretory pathways
i. Clathrin – important for transport between plasma membrane and Golgi, to and from
endosomes in cytoplasm, and receptor-mediated endocytosis
1. Receptors found in clathrin-coated pits, i.e. LDL-receptors or growth factor receptors
ii. COPI: golgi to ER (retrograde)
iii. COPII: ER to golgi (anterograde)
14) Lab Techniques
a. PCR – amplifies DNA molecules in a sample
i. Uses:
1. Make more DNA from small amount
2. Determine if DNA is present (Does it amplify?)
3. Determine the amount of DNA present (how quickly?)
ii. Ingredients:
1. Sample of DNA, DNA polymerase, Primer (Single-stranded DNA segment complementary
to DNA being evaluated), Nucleotides
iii. Technique – process is repeated in cycles that each generate more DNA
1. Heat Sample (DNA denatures into single strands)
2. Cool Sample (primer anneals complementary DNA)
3. Warm Sample (DNA polymerase elongates from primer)
iv. Real time PCR – Quantitative PCR
1. PCR done in presence of fluorescent dye – the amount of dye is proportional to amount
of DNA and is detected as PCR is ongoing
a. More DNA = more fluorescence
b. Rapid increase in florescence = more DNA in sample
v. Clinical Uses
1. Herpes simplex virus encephalitis – DNA in CSF
2. HIV Viral Load
a. Uses reverse transcriptase to make cDNA
b. Amplification of cDNA
c. Amount of cDNA produced over time indicates viral load
d. Standard tool for monitoring viral load
b. Blots
i. Southern – DNA
1. Uses probe to identify presence of DNA in sample
a. Probe: single stranded DNA molecule that carries radioactive or chemical
markers and binds complementary sequences in a sample (Probe can be called
cDNA, binding referred to as hybridization). Once the probe has bound, markers
reveal DNA in the sample
2. Technique
a. Step 1 – Add restriction nucleases to a sample of clumped up DNA; restriction
nucleases chop DNA up into smaller fragments
b. Step 2 – Use Gel Electrophoresis to separate the fragments by size; provides
spectrum of all pieces of DNA separated by size
c. Step 3 – Blotting – transfer to filter paper – DNA sticks to filter paper
 d. Step 4 – add probe and wash away unbound probe; bound probe remains on
filter paper; filter paper exposed to film and bound DNA is revealed
i. Done with multiple samples for comparison
3. Clinical Uses
a. Restriction fragment length polymorphisms – technique used to characterize
genes
i. Restriction Nuclease cut DNA at specific base sequences creating
fragments of genes
ii. RFLP analysis involves anlyzeing the fragments – diff genes break to
different length fragments and so are identifiable
iii. Could analyze unknown gene to see the fragments and compare to a
known product; can use them to determine genotype of a patient, for
example
b. Sickle Cell Anemia can be diagnosed using Southern Blotting
i. Normal beta-globin breaks into two fragments, while Sickle cell patients
have only one fragment
ii. Northern – RNA – useful for assessing mRNA levels (gene expression)
1. Same technique as Southern
2. Clinical Use: to identify gen expression – if you can identify an mRNA fragment, you can
see that the gene is being expressed
iii. Western – Protein
1. Same technique but for protein; instead of probe use an antibody for the specific
protein
2. Clinical use: detection of antibodies
a. IgG or IgM in Lyme disease
b. IgG HIV-1
iv. Southwestern Blot combines features of southern and western blots
1. Use: to study DNA-protein interaction; like DNA-binding proteins, especially
transcription factors
2. Technique:
a. Proteins separated by electrophoresis (like in western blot)
b. DNA probe added (southern blot) to see if it binds to the protein fragments
c. Flow Cytometry: analysis of cells as they flow in a liquid through a narrow stream to analyze by size or
present surface proteins
i. Use: Analyze cells by size, granularity, or surface protein
ii. Components: Flow Cytometer creates side scatter and forward scatter
1. Flow cell: Moves cells through machine
2. Laser: to produce light scattered by cells
a. Forward Scatter = size
b. Side Scatter = granularity
3. Photodetector: detects the light scatter
iii. Technique: use flow cytometer to scatter light and measure the scatter, plot the front scatter vs.
the side scatter to identify different populations of cells
iv. Antibody Staining using specific antibodies to surface/intracellular proteins to indicate the
presence of a protein in cells
1. Antibodies are tagged with a unique fluorochrome that is detectable by the flow
cytometer if bound; if detected, indicates the presence of protein in cells
2. Possible with multiple antibodies with unique fluorochrome (i.e. bind different
antibodies to CD4+ and CD8+ cells
 v. Clinical Uses:
1. Fetal maternal hemorrhage
a. Flow cytometry employs monoclonal antibodies to hemoglobin F – if fetal
hemoglobin is detected in the red cells, means they have crossed to maternal
side
2. Paroxysmal nocturnal hemoglobinuria – can be identified with fluorescently-labeled
monoclonal antibodies that bind to GPI anchored proteins (deficient in red cells of these
patients)

d. ELISA – Enzyme Linked Immunosorbent Assay

i. Detects antigens and antibodies (proteins) in serum based on enzymatic color change reaction
ii. Several different variants (direct, indirect, sandwich, competitive)
1. Direct: Enzyme-linked antibody binds DIRECTLY to the antigen
a. add serum to be tested to coat the plate → antigens secure to the surface →
wash away fluid, leaving antigens on the plate
b. Add enzyme-labeled antibody specific to the target antigen, and wash away
unbound antibodies
c. Add substrate that undergoes color change in presence of enzyme – a color
change indicates binding
2. Indirect: Enzyme linked antibodies bind INDIRECTLY to antigen
a. Add serum for analysis
b. Add antibody to antigen of interest (antibody is NOT enzyme linked)
c. Wash away unbound antibody
d. Add enzyme labeled secondary antibody that binds antibody that bind to the
antigen
e. Add substrate and observe color change (if antigen is present)
3. Direct vs. Indirect
a. Direct – time consuming to label antibodies to unique antigens
b. Indirect – Easier to acquire specific antibody (i.e. a mouse antibody) and then
use a generic secondary antibody (i.e. anti mouse antibody)
4. Sandwich: Antigen will end up sandwiched between two antibodies
a. Plates coated with capture antibody
b. Sample added, present antigen binds to antibody
c. Add detecting antibody that binds to antigen
i. Direct: detecting antibody is enzyme linked
ii. Indirect: secondary enzyme-linked antibody added
d. Add substrate and measure color change
e. Sandwich method advantages:
i. High specificity – since need two antibodies it is unlikely to bind wrong
antigen
ii. Works with complex samples and does not require purification
iii. Can use secondary antibody like indirect
5. Competitive
a. Primary antibody incubated with sample so antigen-antibody complexes form;
with more antigen, more binding, and less free antibody to bind the coated
plate
i. Mixture added to antigen coated plates → unbound antibody then
binds antigen on the plates
ii. Wash away antigen-antibody complexes
 iii. Secondary antibody and substrate added
iv. More color change = LESS antigen in the sample
iii. Clinical Uses:
1. HIV antibody detection – use ELISA to identify HIV antibodies in the serum
a. Indirect often used
b. HIV antigen attached to well
c. Sample reacts with antigen-coated plate
d. Secondary antibody: antihuman immunoglobuin with bound enzyme – color
change means HIV antibody present in the sample
2. HIV p24 antigen using sandwich ELISA (though many variants used)
e. DNA Microarrays and FISH
i. DNA Microarray – also called DNA chip or biochip; solid glass, plastic, or silica structure that
contains thousands of DNA sequences
1. Used to test a sample of DNA with fluorescent markers by hybridizing with
complementary bases → a computer detects which probes bind to the sample allowing
for the identification of DNA patterns
2. These have automated the identification of DNA
3. Research uses:
a. Gene Expression – determine which genes are active/inactive (i.e. cancer cells
vs. normal cells)
i. Collect mRNA → cDNA which is tested using microarray
b. Copy Number Variation – some cells have ↓↑ copies of genes/DNA that are
associate with disease
i. Cellular DNA is collected and tested vs. reference sample; results
compare the fluorescence of the sample compared to the normal
reference to understand the difference
c. Single Nucleotide Polymorphisms (SNPs) – genes exist with variations in a single
nucleotide
i. Many SNPs are associated with disease and are preserved within
families
ii. FISH - Fluorescence in situ hybridization
1. Uses fluorescent lighting to ID binding of complimentary DNA segments by binding to a
specific gene site – helps localize to specific chromosomes to see which chromosome
holds a gene
2. Technique
a. Fluroescent DNA probe complementary to the gene of interest is hybridized to
the DNA of interest to see where it binds
b. Often done on cells in metaphase – where cells are arrested and chromosomes
are visible; the chromosomes are fixed to a glass slide
i. DNA probes that match regions of known chromosomes will hybridize
to chromosomes on the slide; Visualize with fluorescence microscopy
3. Clinical Usage – to compare test cells to normal cells in order to locate a gene
a. Microdeletion: no fluorescence of chromosome (i.e. DiGeorge Syndrome)
because probe does not bind due to genetic deletion
b. Translocation: fluorescence on the wrong chromosome
c. Duplication: extra site of fluorescence
15) Cytoskeleton (Microfilaments, Microtubules, Intermediate Filaments)
a. Overview: structural framework within the cytosol; function to maintain cell shape, stabilize cell
attachments, facilitate endo and exocytosis, and promote cell movement
b. Microtubules
i. Structure:
1. straight, hollow tubules made of tubulin
2. 13 protofilaments with a linear arrangement of tubulin dimers of α and β tubulin
subunits linked end to end
3. Polar (β plus end, α minus end)
4. Polymerize at the + end, in the presence of GTP; - end usually affixed to MTOC
ii. Microtubule-associated proteins – stabilize, control growth, and bind microtubules to other
cytoskeletal components and organelles
1. Associated with kinesin and dynein – force generating motor proteins; serve as the
motors for vesicle or organelle movement
a. Kinesin moves towards the + end (toward cell periphery)
b. Dynein moves towards – end (toward center of the cell)
iii. Function: maintain cell shape, aid in transport of macromolecules, vesicles and organelles,
assemble into mitotic spind, assist formation of cilia and flagella
iv. Centrosome (Microtubule Organizing Center (MTOC) and Centrioles)
1. Structure
a. Located near the nucleus, with two centrioles (cylindrical rods)
b. Centriole composed of 9 triplets of microtubules arranged radially
c. Centrioles self-duplicate
2. Function: major microtubules organizing center in the cell
a. Contains γ-tubulin that serves as the starting point for the polymerization of one
microtubule
b. Centrioles help maintain the organization of the centrosome
c. Centrosome duplicates during interphase, separates to form the poles of the
mitotic spindle
c. Microfilaments (actin filaments)
i. Structure:
1. composed of globular actin monomers (G-actin) linked into a double helix (F actin); thin
flexible, abundant
2. Polarity – polymerization at the + end (barbed end) relative to the minus end (pointed
end)
3. Free G actin is bound to ATP, subunit hydrolyzes ATP during binding, releases energy,
and ADP is buried deep in the groove
4. Actin binding proteins associate with G and F actin to accelerate/decelerate
lengthening/shortening + permit branching; certain condition produce "treadmilling"
where the same number of G actin monomers added/removed
5. Abundant at the periphery of the cell where they are anchored to plasma membrane via
intermediary proteins
ii. Function
1. Establish contacts between the cells and the extracellular matric, locomotion of non-
muscle cells, formation of contractile ring (in dividing cells), form rigid core in microvilli,
fold epithelia into tubes during development
d. Intermediate Filaments – population of heterogeneous filaments that are product of at least 70 genes
i. Structure
 1. Monomers (composed of rod-shaped protein with alpha helical structure, central
domain, with N terminal and C terminal) coil together to form dimer, dimers assemble
head to tail, antiparallel, in tetramers – finally, Unit length filaments are 8 tetramers
together
ii. Function: provide mechanical strength to cells, lack polarity, and do not require ATP or GTP for
assembly
e. Cilia and Flagella!!!!

16) Cancer – cells no longer respond to normal restraints on growth, resist apoptosis
a. Difference between normal and cancer cells
i. Accumulation of mutations in several gens results in transformation – cells change morphology,
proliferate, invade tissue, metastasize. Steps:
1. Mutation in proto-oncogene OR Tumor-suppressor gene
2. Mutated cell proliferates
3. Multiple mutation accumulated
4. Cells invade surrounding tissue and blood vessels resulting in metastase
ii. Oncogenes – a gene that causes cancer – activated by a gain of function mutation that mutates
the a gnee that controls normal growth and division
1. Proto-oncogene – corresponding “normal” cellular gene
2. Activation of oncogene by
a. DNA Damage (altered base sequence) →
i. Nucleotide excision repair errors → xeroderma pigmentosum
b. Mutations in Signal transduction cascades (i.e. growth factors, signal
transduction proteins, or transcriptions factors)
3. Gain of function mutations:
a. Altered regulation of expression
b. Translocation (CML)
c. Amplification or over expression
4. Oncogenes and the cell cyle
a. Cyclins and Cyclin dependent kinase – control progression from one phase of
cell cycle to another
b. Cyclin-CDK complex regulated by phosphorylation by inhibitory proteins that
slow down the Cell Cycle
c. G1/S transition regulated by: Cd4, cd6, cyclin D, Rb, E2F transcription factors
i. Cdk4 and cdk6 bind cyclin D → activation, Rb is phosphorylated →
dissociates from E2F, allowing new gene transcription

Class Proto-Oncogene Activation Location Disease

G-Protein Ras Point Mutation Cytoplasm Multiple Cancer
(i.e. leukemias)
Serine-Threonine Raf Overexpression Cytoplasm Myeloid leukemia
kinase
Tyrosine Kinase Abl Translocation Cytoplasm – CML
chromosomes 9 and
22
src Overexpression Cytoplasm Colon carcinoma
Transcription Factor Myc Translocation/amplification Nucleus – Burkitt Lymphoma
chromosome 8 and
14
 iii. Tumor Suppressor genes – loss of function mutation leads to uncontrolled cell growth –
recessive oncogenes (loss of both alleles is required)
1. Retinoblastoma gene (rb) mutation alters G1/S transition allowing cells to cycle when
they should not be cycling; sporadic (not genetic) retinoblastoma acquires two specific
mutations
2. P53 – TF that regulates cell cycle and apoptosis; loss of function is found in more than
50% of tumors; p53 stops the cell cycle when DNA damage is found
a. If DNA damage cannot be repaired, p53 activates genes involved in apoptosis
3. Ras – loses GTPase activity and becomes constitutively activated
iv. Clinical Correlates
1. Chronic Myelogenous Leukemia (CML) – translocation between chromosome 9 and 22
(Philadelphia chromosome)
a. Fusion protein (chimeric) – bcr-abl – abl is tyrosine kinase under bcr gene
control that leads to inappropriate expression, cell proliferation, and inhibition
of differentiation
b. Treated with Gleevec (imatinib) – inhibits activity of the specific kinase activity
2. Burkitt Lymphoma – translocation of proto-oncogen myc, a transcription factor –
constitutive activity of promoter leads to inappropriate cell growth – common
translocation between chromosome 8 and 14
a. Translocation linked to EBV in 95% of case
v. DNA Repair enzyme mutation – Enzymes in DNA repair are Tumor Suppressors as loss of
function leads to increased probability of mutation
1. BRCA1 and BRCA2 (associated with breast cancer) – repair single and double strand
breaks – since at risk individuals inherited a mutated gene a mutation in the second will
cause cancer
2. Li-Fraumeni – due to inheritance of mutated p53 (the guardian of the genome), results
in the development of multiple tumors as the normal allele lose its function
3. Ataxia Telangiectasia
b. Metastases
c. Possible Cell Changes
d. Oncogenic Microbes
i. Three RNA retroviruses have been associated with human cancer
ii. DNA viruses can lead to transformation
1. EBV encodes a Bcl-2-like factor and antagonizes apoptosis in infected cells
a. Bcl-2 is ANTI apoptotic
First Aid for Basic Science 39-59, 169-178

I) DNA Structure
a.

First Aid Usmle 1- 32-42, 48-51