Cell bio exam 1

Spontaneous Generation

non-living objects can give rise to living organisms

Francisco Redi

Disproved spontaneous generation by showing that maggots on decaying meat came from fly eggs, not from the meat itself. Maggots on meat came from fly eggs (open container), while maggots did not grow into flies in the sealed container.

Jansen

1595, created the first compound microscope

Robert Hooke

first described cells

Antony van Leeuwenhoek

first to see prokaryotic bacteria and living protozoa/sperm/bacteria using the single lens microscope

Cell theory

1. cells are fundamental units of life

2. all living things are made of cells

3. new cells only arise from pre existing cells

Cells are…

small (0.1um-1mm)

membrane bound

packed with chemicals and macromolecules (gel-like)

can make copies of themselves

DYNAMIC

Molecular commonalities

DNA is genetic material

same sugars and amino acids

ATP for energy

have membrane proteins

phospholipid membranes

Central dogma

DNA → mRNA → protein

Prokaryotes

Bacteria and archaea with no nucleus or organelles, unicellular, small (0.1-10um), transcription and translation not physically separate

Eukaryotes

Animals/plants/fungi/protists that have nuclei and organelles, multicellular, large (5um-1mm), transcription and translation are physically separated

Where does eukaryotic transcription occur?

nucleus

Where does eukaryotic translation occur?

cytoplasm

Where do transcription and translation occur in prokaryotes?

cytosol

Nucleoid

where prokaryotic DNA is supercoiled, usually in a singular, circular chromosome

lysozyme

cleaves peptidoglycan in bacterial cell walls (cleaves the beta 1,4 glycosidic bond between N-acetyl muramic acid NAM and N-acetylglucosamine NAG sugars)

Organelles function in eukaryotic cells

1. compartmentalization of materials and reactions → regulation and efficiency (make or break things) and storage

2. cell/tissue differentiation: different cells have different needs/functions

3. SA:V

high SA:V will have…. rate of exchange of molecules with the environment relative to the internal volume than a cell with a …. SA:V

higher, low

Problems with low SA:V

1. slow exchange of materials with environment relative to volume (solution: endocytosis and exocytosis)

2. many enzymatic reactions occur on/at/in membranes

origin of eukaryotic cells

anaerobic archaea uptook aerobic proteobacterium = symbiosis of aerobic respiration

archaea are more closely related to eukarya than bacteria

true

mitochondrial endosymbiosis

all extant eukaryotes have mitochondria

plastid endosymbiosis

chloroplast is remnant cyanobacterium that allows plants to do photosynthesis

archael links

TACK archaea have proteins in common with eukaryotes, suggesting the first eukaryotic cells derived from TACK archaea

Some traits are common to all cells

ribosomes, phospholipids, nucleic acids, core metabolism

Some traits were acquired during cell evolution of specific brances and are common only to particular branches

TACK archaea: proteosome, core histones, N-linked glycans

Eukarya: genome expansion, gene duplication, multicellularity

Transition analyses

comparing metabolic pathways, cellular structures, organelles

Hypothetical model for eukaryotic cell origin

1. TACK archaea lose cell wall

2. actin cytoskeleton is altered for phagocytosis

3. phagocytosis of numerous bacteria by archaea with transfer of some DNA to nucleoid (horizontal gene transfer)

4. development of membrane around nucleoid toform nucleus and uptake of bacterial endosymbiont that maintains independence as mitochondrion

5. mitochondria multiply in primitive eukaryotic cells

Gene families

Homologs

genes that are related by descent, with similar sequences and functions

ex. orthologs (different species, same function) AND paralogs (same species, same function)

Paralogs

arise by a gene duplication event within a single genome, go on to have many individual organisms with multiple copies of genes with similar sequences/functions

same species, same function

Orthologs

different species, same function

Evolution of genes and genomes

intragenic mutation

gene duplication (meiosis) → functional divergence (one can develop mutations)

DNA segment shuffling

horizontal DNA transfer (antibiotic resistant genes or endosymbiosis)

Cell lysis

1. put tissue in buffer at specific pH

2. disrupt tissue w/mortar and pestle or blender or homogenizer

French press

put cells under high pressure → rapid release of pressure

Sonification

probe vibrates very rapidly

cavitation

vapor filled cavities form and collapse to lyse cells

Detergent lysis

dissolves cell membrane (disadvantage: can denature protein, doesn’t work on cells with cell wall)

Osmotic shock

put cells in hypotonic solution (water flows in and cell lyses)

Cell fractionation (centrifugation)

differential, density gradient

differential centrifugation

low spins, separation based on size/density of subcellular structures (pellet and supernatent); separate cytosol from rest of cell

density gradient ultracentrifugation

high speed

velocity sedimentation

sucrose gradient and lysate on top → centrifuge → small less dense things on top, larger denser things near bottom

equilibrium sedimentation

lysate is mixed into gradient → centrifuge → cell components migrate according to density until neutral density is reacted

Protein purification

chromatography

Gel filtration (size exclusion)

separation by SIZE

solvent flows through porous beads and small proteins get stuck in beads (big proteins elute first)

Ion exchange

separation based on CHARGE

any protein can be positively or negatively charged, depends on buffer pH and isoelectric point pI

isoelectric point pI

pH where protein is neutral (no net charge)

pH > pI

protein is negatively charged (fewer protons)

pH < pI

protein is positively charged

Anion exchange

positively charged beads → negatively charge molecules interact while positively charged molecules flow through (positively charged proteins elute first)

increase salt concentration

disrupt proteins that interact with positive matrix

How does phosphorylation affect charge?

adds negative charge to proteins

Cation exchange

uses negatively charged matrix so positively charged molecules interact

Affinity chromatography

stationary phase has something covalently attached; protein sticks to something specifically while other proteins flow through (low affinity); eluting solution contains something that compete for binding

SDS page

determine purity

vertical polyacrylamide gel electrophoresis (negative cathode → positive anode) → negatively charged proteins migrate towards positive pole (bottom) → small things move quickly, slow large things move slowly

SDS

sodium dodecyl sulfate; strong anionic detergent; denatures proteins AND gives them net negative charge; all proteins get same mass/charge ratio, separation on SIZE only

What does SDS PAGE tell us?

size, purity, how much (darker band = more)

beta mercaptoethanol

breaks disulfide bonds

2D SDS PAGE

first dimension is isoelectric focusing (proteins separated by electrophoresis in gel w/pH gradient, proteins migrate until they reach a place w/no net charge pI)

second dimension is SDS PAGE

How does phosphorylation affect isoelectric point (pI)?

pI lowers because phosphorylation adds more negative charge therefore more acidic conditions (more H+ ions) are needed to reach pI

Antibodies

produced in B cells in response to antigens

polyclonal antibodies

recognized different epitopes of the same antigen

monoclonal antibodies

normal, somatic cells can be fused with tumor cells to form a heterokaryon; recognize a single epitope

Western Blotting

detection of a protein on a membrane after SDS PAGE

primary antibodies

recognize protein of interest (produced in one species)

secondary antibodies

recognize first antibody (produced in another species and recognizes the Fc region of the first species’ IgG)

contains label (enzyme)

2 identical SDS PAGE gels are run

one gel stained for total protein, one gel blotted onto membrane and investigated for single protein w/an antibody

Protein interaction methods

use genetic engineering to add “epitope” tag (extra protein [fusion protein] sequence that is translated w/gene for protein)

Tags can be used for…

1. affinity purification and ‘pull down’ assays

2. Immunoprecipitation assays

3. detection (Western blot, microscopy)

Pull down assays

depends on affinity chromatography → protein binds to column via glutathione S transferase as “bait” ; proteins from cell lysate are added “prey” ; bait and prey complexes eluted from column together

Co-immunoprecipitation

harvest and lyse cells/tissue

inert bead w/specific antibody will bind to protein (+ other interacting proteins)

elute protein complex from beads

Chromatin immunoprecipitation (ChIP)

protein DNA interactions

1. Cross link transcription factors of proteins (w/formaldehyde)

2. lyse cells

3. break DNA into small fragments

4. use antibodies against protein

5. protein is purified along w/the bound DNA

6. amplify the purified DNA (DNA corresponds to transcription regulators)

Resolution

smallest distance b/w two points in an object that is still perceived as separate in the image

highest resolution of human eye

0.2mm (200 um)

highest resolution of light microscope

200 nm (0.0002 mm)

highest resolution of electron microscope

0.2 nm

standard compound light microscopes

upright

inverted: cells in petri dishes → bottom of dish is ‘cover slip’, image from bottom

effective magnification

eyepiece (10x) x objective lens (up to 100x)

Cells in tissues

fix: heat w/chemicals to maintain morphology

section: cut into thin slices w/microtome (embed in wax or plastic) → slice think sections

place section on slide (maybe stain)

image

Problem of light microscopes

light microscope have max. resolution of 0.2 um (200nm) and many cellular structures are smaller than this

Solution

use electrons (very short wavelengths), refracted by smaller structures

Transmission electron microscopy (TEM)

very high resolution, 2nm

can only image fixed and sectioned samples

similar to compound light

electrons pass through very thin slice of specimen

Scanning electron microscopy (SEM)

very high resolution, 3-20nm

image intact tissues/cells but can’t see inside (unless broken open)

similar to dissecting microscope; electrons bounce off specimen

look at cell surface (vs. slice)

Eukaryotic chromosome structure

multiple linear chromosomes per nucleus, each chromosome is a single DNA molecule that maintains genetic info and replicates genetic info

Prokaryotic chromosome structure

one circular chromosome (nucleoid) and plasmids (extra chromosomal DNA)

telomeres

protective caps on chromosome, get shorter every time cell divides

centromere

mostly heterochromatin, required for segregation by kinetochores in M-phase

Nuclear envelope

boundary of nucleus; 2 lipid bilayers (inner and outer, contiguous with ER)

nuclear pore

gated, control entry and exit of macromolecules (RNA and proteins)

nucleolus

where ribosomes are made; transcription of rRNAs by RNA polymerase I and ribosome assembly

nuclear lamina

meshwork of supportive proteins

Chromosomes are made from chromatin

DNA and associated proteins

nucleosome

core unit, DNA wraps around histone heterooctamer core (~1.6x)

linker DNA

variable length, depends on chromatin state

nuclease-protection assays

1. isolate nuclei with DNase I (naked DNA is cut)

2. treat with high conc. of salt to separate histone octamer and DNA

3. DNA gel electrophoresis - big DNA is more negatively charged and stays near the top, while positively charged small DNA moves to the bottom

Histones

heterooctamer- 2 of each protein subunit (H2A, H2B, H3, H4)

subunits are small, basic (lysine and arginine) with a positive charge, and have N-terminal tails that can be modified

Histone H1

not part of core, helps compact DNA in heterochromatin (mitotic chromosomes)

DNA folding

naked DNA —> nucleosome beads on a string —> 30nm fiber of chromatin —> loops —> mitotic chromosomes

Euchromatin

loose, long linker DNA between nucleosomes = express genes