B1 Nitzan

Prok. = nucleoid-irregularly shaped region contains most/all gen material

No cytoskeleton/organelles, 1-5 x 10^6 pdb (vs 10^7-9)

Biggest cell = eggs

Protozoa = unicell euk.

Prok. = archaebacteria / eubacteria

Archae = extreme envi :

1 – methanogens = anaerobic, produce methane, swamps, sewer, gut (cow)

2 – thermoacidophiles = high temp and low pH

3 – Halophiles = salty ++ (dead sea)

Eubacteria = include bacteria, spherical, rod-shaped, spiral (diam 1-10pm)

Experimental models:

1 – Prok. = E. coli, simple genome, easy growth, div 20-60mn

2 – Euk. = Saccharomyces cerevisiae, yeast, simplest euk., 16 chr., cytoskl/organelles

3 – Drosophila melanogaster = replicates +, easy growth

4 – Xenopus laevis = early dev, large eggs, dev outside body

Microscopes

LM = wave length 0,4-0,7um (purple-red), living cells not visible unless phase contrast microscope

EM = 1000x (0.1nm), wavelength decreases as its velocity increases (0,004nm), 100.000V, aberration
of the electron lens, heavy metal salts fixation needed, staining with dense material

Dark field microscope : side light, cell = bright objet on dark ground
Fluorescence microscope : fluo molecule absorb light at a wavelength and emits it at another longer
WL, filter allows only the emited WL to pass

Confocal microscope : fluo microscope + electron image analysis = 3D picture

Cell membrane

Lipids through hydrophobic barrier, hydrophilic via protein specialized- transport, recognition/comms
via carbohydrates prot, energy conversion/enzymatic reactions via prots

Phospholipids = amphipathic = 2 fatty acid chain = phosphate containing head

Lipids = 50% of most animal cell mb, 4 types :

Phosphatidylserine = neg, only inner, if outer = macrophage induction (phago dead cell)

Phosphatidylethanolamine = mostly inner

Phosphatidylcholine/sphingomyelin = mostly outer

Also contains glycolipids and cholesterol

Individual mols (lipids and prots) are free to rotate, move laterally, flip/flop

Chol makes bilayer less fluid (fluid mosaic model)

Chol and sphingolipids (sphingomyelin + glycolipid) form discrete lipid rafts (small semi solid patch),
necessary for endocytosis

Extracell part of mb prots are generally glycosylated, same for carbs part of glycolips

Forms glycocalyx = carbs coat, formed by oligosaccharides of glycolips and transmb glycoprots,
protects the cell surface, also oligosaccharide serve as markers for cell-cell interactions

Mb prots :

Trans/integral : embedded directly within the LBL

Peripheral = bound to either face

TRANSPORT :

Passive = no energy, co gradient :

Simple diff = small and nonpolar mols (O2, CO2)

Facilitated diff = large/polar/charged via integral prots :

- channels prots = pores, free diff of any mols of appropriate size/charge

- carrier prots = bind specific prots, conformational change (uniport, symport, antiport)

Active = against gradient, energy = ATP/ion gradient

Na-K pump = ATP hydrolysis, antiport, 25% of cell ATP cons

Reg cell vol through osmotic effect, nerve impulse, assists in active transport of mols (glucose,
symport), secondary active pump. Indirect hydrolysis of ATP, usage of energy concentration gradient
created by primary active transport

ABC (ATP binding cassette) transporters : family of mb transp with ATP binding cassette

MDR prot (multidrug res) = remove toxic forign compounds, found in cancer cells

CFTR (cystic fibrosis transmb regulator) = Cl- channel in epith cells, CF = often single point mut,
interferes with prot folding + Cl- transp, recessive, thick mucus, respi + gastroint epith, CL- remains
inside cell, forms thick mucus with water, breathing problems, infections, sealed pancreas ducts,
salty sweat (diag)

RBC mb = best study cells (no nucl/organelles -> mb/mb prots easily isolated), 52% prots, 40% lips,
8% carbs, pale empty cells = RBC/white ghosts = hemolysis, disease

SDS

SDS-PAGE = separation of charged mols by their charge, sodium dodecyl sulfate identifies 15 diff
prots

RBC cytoskel = spectrin ++, in tetramers, bind with actin for spectrin-actin network (cortical cytoskel),
linked to mb via ankyrin wich binds to transmb prot band 3. Another link is prot 4.1 (spectrin/actin
junction – cytoplasmic domain of glycophorin), mut spectrin = spherocytosis, sphere, less surface
tension, less flexible, band 3 defficiency (transp passage bicarb HCO3- and chloride ion Cl-) across
RBC mb, also spherocytosis

Dystrophin = spectrin related prot, actin – muscl cell mb, protects muscle cells during contraction,
mut = Duchenne muscular dystrophy = boys + (X), muscle degen/weakness

Cell junctions = epith cells ++

1 – Adhesion junction = specific between same tissue cell

2 – Tight junction = occludine prots, seal epith sheets, separate apical/basolateral domains, found in
BBB (blood brain barrier)

3 – Gap junctions = connects adjacent cells cytoplasm, ion/small mols diffuse freely, 6 connexins
(transmb prots), pore = connexon, allows mb potential to pass from cell to cell (cardiac muscle),
cancer cells lack gap

4 – Desmosome junctions = tight junctions between epith/muscle cell, attached to intermediate fil of
keratin in cytoplasm, keeps tissues tight, pemphigus = autoimm disease against desmosomes

Intracell compartments – Organelles

Nucleus = dble layer mb, comms via highly selective specialized pores, also nucleolus = region of
highly densified ribosomal RNA which aids in the transcription process

Ribosomes = 5-10M/division, on rough ER after initiation of prots synthesis/in cytoplasm, made of

two components : small ribosomals sunits for reading RNA, large subunits wich joins AA to make
polypeptide chain

RER = network of tubules and sacs enclosed in a mb, continuous with nuclear mb, covered with
ribosomes, signal sequence in newly formed polypep directs engaged rib towards ER mb (if no signal,
cytoplasm), signal seq binds to SRP (Signal recognition particle) that leads him to SRP receptor near
translocator channel prot that permits translocation to the ER lumen

Secretory Pathway = RER – Golgi – secretory vesicles – cell exterior

Prot Glycosylation : addition of carbs, vast majority of ER synthesized pots are glycoprots, as soon as
a polypep enters, a common oligosacc synthesized in the ER on a lipid carrier (dolichol phosphate) is
transferred on an Asn

Prot Folding = polypep chain enters unfolded, is folded in ER lumen, then transferred folded and
partially glycosylated in vesicle to Golgi

Golgi = up to 100/cell, membrane enclosed sacs (cisternae) and associated vesicles, factory where
prots from ER are further glycosylated, sorted (via addition of different carbs) and packed for
transport to : lysosome, plasma mb, secretion; they go from cis to trans face, RER is in constant
contact with golgi via 2 coatomer coated vesicles : COP II coated vesicles (ER to Golgi), COP I (along
Golgi, Golgi to ER)

Lysosome = enzymes produced through ER/Golgi, break down polymers (prots, AN, carbs, lips), pH 5,
endosome = mb organelle in animal cell, carries material ingested via endocytosis, pass majority to
lysosome (early endosome -lowers pH-> late endosome -fuses with lys. enzyme laden vesicle->
lysosome)

Endocytosis : Phagocytosis = large particles, Pinocytosis = solutes/single molecules (prots), Receptor

mediated endocytosis = select., macromols, receptors usually located in clathrin-coated pits, trash
released in vesicles via exocytosis
Uptake of cholesterol = LDL particles = chols surrounded by phospholip monolaye + Apo-B prot,
familial hypercholesterolemia = autosomal dominant, no/changed LDL receptors, accumulation on
arterial walls, PA +, atherosclerosis

Uptake of transferrin = iron from digestive syst to liver via transferrin, apotransferrin + 2 Fe3+ =
ferrotransferrin

Lysosomes diseases : tay sachs = gen, destruction of nerve cells and spinal chord; gaucher =
glucocerebroside accumulated in cells and organs; silicosis/asbestosis = lung disease; rheumatoid
arthritis = autoimm, joints; gout = inflammatory arthritis

SER = cell with lipid metabolism ++, netlike labyrinth of tubules, continuous with RER, functions :

Lipid biosynthesis = enzyme for synth located on cytoplasm face of ER mb, enzyme flippase transfers
synthesized lips to inner layer if they remain in ER, lips are transferred to other organelles bound to
prots

CA2+ storage = sarcoplasmic reticulum = specialized SER in muscle cell, release/reuptake of Ca

triggers contraction/relaxation

Prod of cytochrome P450 enzyme = catalyzes a series of reaction to make water insoluble
drugs/metabolites (alcohol) soluble enough to be excreted in urine

Peroxisomes = small, single mb organelle, 500/cell, 50+ enzymes, carry out oxidation reaction that
produce toxic hydrogen peroxide H2O2, decomposed by catalase in 02 and H20, contains oxidative
enzyme that uses oxygen for degradation of AA, purines, methanol, fatty acids; involved in chol and
dolichol biosynthesis; perox prot cyto synth, perox phospholips ER synth, additional entry of both
leads to growth then division; diseases : Zellweger syndrome = no entry of perox prots in perox
(empty), early death; Peroxisomal protein signal sequence mut = Alanine glyoxylate
aminotransferase (AGT) enzyme has a changed signal sequence, directed to mitochondria instead, no
function, formation of kidney stone

Cytoskeleton : microfilament (actin) + microtubules + intermediate filaments

Microfilaments : major CS prot in most cells, polymerizes to form microfilaments (e.g myosin),
beneath mb + for shape/movement, myosin = prototype of motor-a protein that converts ATP to
movement (muscle); globular G-actin binds tightly and polarly into filaments, used ++ by bacteria

Microtubules : dimer of α and β tubulin, polym. into microtubules made of 13 linear protofil. around
a hollow core, 25nm diameter, polar, continuous (dis)assembly from – α end to + β end

Intracell. transports (ves. and orgl., kinesin/dynein), mitosis, cilia, flagella; centrosome = 2 centrioles
(each 9 triplets), near nucleus during interphase, replication before cell div. (cancer cell = incorrect
mitosis = 4 centrosomes); Cillia/Flagella : 9 doublets + 2 in the middle, dynein generates mov.; basal
body : cylindrical orgl., base of flagellum/cilium in the cytoplasm, same structure as centriole

Intermediate filament : only structural, variety of prots in diff. cells, 6 groups :

Keratins : epith, 15 diff. prots, epidermolysis bullosa = mut. autodom, blistering of the skin at light
stress

Vimentins : fibroblasts, SMC, WBC, attached to microtubules for shape

Neurofilament : in mature neurons along axon, assemble the nerve cell CS with longitudinal
microtubules
Lamins : nuclear, inner mb of nuclear envelope, important for mitosis, disassemble if phosphorylated

Mitochondria : own DNA, encodes small part of its tRNAs, rRNAs and some mitochondrial prots

Structure : double mb with folds (cristae) around matrix, outer mb = porins + (aqueous for mols <
5000 daltons), mitochondrial lipid synthesis / conversion enzymes ; inner mb = cardiolipin + (double
phospholip. with 4 fatty acid chains), impermeable to ion, transport prots and ATP synthase ; matrix =
hundreds of enzymes, ribosomes, t/r/m/mtRNA

Respiratory chain : C6H12O6 + 6O2 -> 6CO2 + 6H20 + energy (36-40% efficiency)

Glycolysis occurs in the cytosol (glucose -> pyruvate), consumes energy

Glucose + 2 (ADP + Pi) + 2 NAD+ --> 2 Pyruvate + 2 ATP + 2 NADH

If absence of O2/mitochondria, fermentation instead (pyruvate -> lactate), Cori cycle (acid lactate
moves to liver to be converted in glucose and etc)

Oxydation of pyruvate into acetyl CoA in the mitochondria : Pyruvate + CoA + NAD+ → Acetyl-CoA +
NADH + CO2(x2)

Citric acid cycle (Kreb) = acetyl CoA oxidated to C02, reduction of NAD+ and FAD to NADH and FADH2
: Acetyl CoA + ADP + Pu + 3NAD+ + FAD -> 2CO2 + ATP + 3NADH + FADH2
Electron transport chain / Oxidative phosphorylation : high energy e- from NADH(2E-) and FADH2 are
transferred through a series of carriers on inner mb, energy from these transfers is used to pump out
H+, H+ comes back through ATP synthase and the energy is used for ADP + Pi -> ATP

10NADH + 2FADH2 + O2 + ∼ 32(ADP + Pi) -> H2O + ∼32ATP + 10NADH+ + 2FAD

In newborn mammals : brown fat tissue, rich in mitochondria with thermogenin that uses proton
flow to generate heat

Endosymbiotic origin theory for mitochondria and chloroplast, same structures and sensible to same
antibio

Multiple circular DNA coding for 13 prots, 2 rRNA (12S, 16S), 22 tRNA, 16 mRNA ; not sufficient for all
DNA activity

Double mb complicates trans., different target signals for different compartment, most studied TS
are amino-terminal presequences, electrochemical gradient drives the prot to the matrix (presequ +,
inside -), HSP 70 keeps prot unfolded, HSP 60 folds prot properly

Mutations and diseases :

1 – Respiratory chain loses 2-3% electrons during their transfer to oxygen -> free radicals -> damages
mtDNA -> mutation

2 – mtDNA maternally inherited -> hundreds of mitochondrial diseases like Leber Hereditary Optic
Neuropathy (LHON) (visual loss, mitochondrial disorder, young males +, mutation of mitochondrial
DNA)
Central Dogma

DNA : sugar phosphate backbone with a nitrogenous base (purine : 2 rings, pyrimidine : 1 ring) in
keto tautomeric held together by H-bonds

Chargaff : ≈ amount of A and T (2 bonds), and of C and G (3 bonds)

Nucleoside = base + sugar, nucleotide are – charged on phosphate group (polyanion)

Watson and Crick : X-ray diffraction taken by Rosalind Franklin and Maurice Wilkins

DNA denaturation : 100°C / pH > 13

Hybridization : 65°C for a prolonged time (works for DNA, RNA and crossed)

Forms of DNA :

A-DNA : double helical, 11 residues/turn, base pairs tilted 20°

B-DNA : classical, 10 residues/turn, perpendicular

Z-DNA : left handed double helical, 12 residues/turn, Alexander Rich, spontaneously ?

DNA replication : multiple origins, bidirectional, 5’ -> 3’, DNA polymerase can’t initiate (≠primases),
Okazaki fragments (1000-3000 pdb)

(dNMP)n + dNTP -DNA polymerase-> (dNMP)n+1 + PPi

Helicase unwind DNA via ATP hydrolysis, single-stranded DNA-binding protein stabilizes it (SSB),
primer (short RNA strand) serves as starting point, later removed from lagging strand

In prok. DNA polymerase III is the major responsible of replication, polymerase I/II/III also act as
exonuclease in both direction, removing primers

In euk. DNA polymerase α/δ/ε (γ in mitochondria) take care of replication, RNA is removed by RNase
H and 5’ to 3’ exonuclease, later filled by polymerase δ and joined by ligase
Trancription

Euk. -> 3 RNA polymerase :

RNA polymerase 1 = nucleolus, rRNA

RNA polymerase 2 = nucleus, mRNA

RNA polymerase 3 = nucleus, tRNA

Promoter region + RNA polym. + transcription factors (majority of gene expression control)

Binding -> DNA open (transcription bubble)

TBP-TATA binding protein = general transcription factor, allows other TF and eventually RNA polym.
to bind

TAFs-TBP associated factors

TFII[x] general transcription factor

Terminator sequence

Basal transcription apparatus = RNA polym. + general factors needed such as :

helix-turn-helix

steroid receptors

zinc fingers

leucine zippers
helix-loop-helix

pre-mRNA to mRNA processing : 5’ guanin cap + 3’ polyadenine tail, 150-200 (both protect the
transcript and help trans. to cyt. rib.)

Splicesomes: 5 types of snRNA + 6-10 prots = SNRNPs (small nuclear ribonucleoprotein), central role
in splicing, intron out, exon stays

Translation

mRNAs read from 5’ to 3’, polyp. chains synthesized from amino to carboxy end

tRNA = adaptors between mRNA / AA incorporated into the prot, L shape, end in CCA-adenosine
ribose-AA 3’, anticodon on the other side of tRNA recognises and binds to codon, AA/tRNA binding
mediated by aminoacyl tRNA synthetase

Stop codons : UAA, UAG, UGA

AUG -> Met

Nucleus

Inner/outer mb joined at nuclear pore complex (50 ≠ nucleoporins, 3-4k pores/cell), bilayer
permeable to small nonpolar mols

Nuclear lamina : structural, 60-80 kDa of lamins (IF)

bidirectional pore traffic :

in : histones, DNA/RNA polym., TF, splicing factor, rib. prots

out : mRNA, rib. subunits, tRNA

Imported prot needs nuclear localization signal (NLS) AA sequence that binds to nuclear transport
receptor (karyopherin +, importin/exportin), ATP/GDP dependent

Some importins (Kap, β) need an adapter karyopherin (Kap α), some are importin/exportin
depending on the protein

Same for export with nuclear export signal In contrast to m/t/rRNAs that function in the cytoplasm,
many small RNAs (snRNAs, snoRNAs) function in the RNA processing of the nucleus ; exported
snRNAs + prots = snRNPS that return to the nucleus

Chromatin becomes highly condensed during mitosis to form the compact metaphase chromosomes
that are distributed to daughter nuclei

During interphase, heterochromatin stays condensed and inactive contrary to euchromatin ;

constitutive heterochr. = DNA not generally transcribed, facultative heterochrom. = DNA transcribed
in other cell types

Nucleolus : site of rRNA transcription and rib. assembly, genes transcribed by RNA polym. I/III, each
gene is a single transcription unit containing the 18s, 5.8S and 28s rRNAs separated by transcribed
spacers ; the genes themselves separated by nontranscribed spacers ; RNA polym. I = 5.8S, 18S, 28S
(200 gene copies) ; RNA polym. III = 5S rRNA (2000 gene copies) ; imported rib. prot + rRNA in
nucleolus = rib. subunits, then exported
2m of DNA into 5-10um nucleus -> chromatin = DNA + protein, histones + (H1/2A/2B/3/4),
nucleosome = DNA + histone octamer

Condensation of chromatin :

DNA folding around prots – nucleofilaments = 7x shorter

solenoid = 40x

filament in loops

chromatid = 50000x shorter than nucleofilament

Human chromosome :

metacentric : middle centromere

submetacentric : near middle, possible L-shape

acrocentric : near-end centromere, chr. 13, 14, 15, 21, 22 ; satellite chromosome = beside
centromere have a segment separated from main body similarly ; telocentric : centromere at the
end, not found in human

To identify chr. : size, banding pattern, centromere position

G-banding : dark = heterochropatin, late-replicated and AT rich ; light = euchromatin, early, GC rich

R-banding : opposite

C-banding : treated with acid and base, centromere stained

Q-banding : fluorescent dye (quinacrine)

Cell culture permitted polio vaccine

Primary culture : directly from organism tissue, allow proliferation until telomere loss causes
structure unfolding ; cell may detect uncapping and stop growing, enter senescence or self-destruct

Many aging-related disease are linked to short telomere

Established cell lines : grow indefinitely, useful for long term research (stem cells, tumor cells)

Two main growth conditions : monolayer (adherent structure) or free-floating (suspension) ; usually
pick the closest to physio conditions

PCR : thermal cycling = DNA melting, DNA primer + polym. + free nucleotides etc