cell bio 1

adapt to environmental conditions

receptor to turn it on

GTP-binding proteins

make second messenger cAMP

protein kinases

phosphorylate and active enzymes

to control molecular composition, growth and differentiation

check for DNA nicks, check for favorable environmental conditions, check for damaged or unduplicated DNA, check for chromosome attachment to mitotic spindle

archaea, bacteria, and eucarya

• occurred about 3.6bya; modern gene sequences are the only surviving evidence
• simple chemicals, simple RNA's that can store info, complex RNAs with catalytic activity, self replication of catalytic RNAs:
• -DNA copies of genetic info
• -encapsulation of nucleic acids in lipid membrane
• -ribosomes synthesize proteins, which dominate cellular catalysts

identical genes diverge from a common ancestor due to a mutation (sister lineages)

diverged as a result of mutations in sister lineages

proteins with same functions but found in different species

• fusion theory: archaea and bacterium are fused and genomes are merged
• engulfment theory: archaea engulfs the bacterium and the transfer of most bacterial genes to Archaea host

prokaryotic extracellular digestive system, fusion of two prokaryotes creates the origin of eukaryote with mitochondria, formation of intracellular digestive system in early eukaryote, formation of membrane organelle and nuclear envelope

compartmentalization, scaffold for reactions, selective permeability barrier, transport, signal transduction, intercelluar interactions, energy transduction. 

• membrane proteins have transmembrane domains where the hydrophobic faces of alpha helices and beta sheets associate with membrane lipids
• beta sheets can form pores with low selectivity

• photobleaching
• laser trap (immobile proteins do not diffuse freely and particles attached resist displacement

using nonionic detergents

integral proteins can be extracted with nondenaturing detergents, peripheral proteins can be extracted using high salt (to disrupt protein-protein interactions

• scaffold for signal transduction
• skeletal support
• cell-cell communication and interaction

lateral diffusion rates in membrane

• def of ?
• inventory of parts
• concentration (biochm, microscopy)
• partners (biochm, genetics)
• rate and equilibrium constants
• biochemical reconsitition
• mathematical model
• physiological tests

• Xenopus egg
• most other cells
• yeast
• bacteria

• You don't have to kill the cell in TEM, but you do in brightfield
• light microscope is limited by point spread function

computational method to remove out of focus light from micrographs; take series of laser sections through a cell and use computers to stack images looking at 3-D image of cell, use that go from blurry to high resolution image

absorption of a photon raises an electron to an excited state; a longer wavelength photon is emitted when the electron falls back to the ground state

needs to be microinjected

be injected or fused to cell

^{in situ hybridization in fixed cells}

antibody isolated from animals immunized with target molecule

• cDNA from jellyfish can be fused to genes for many proteins and expressed in live cells
• variants are yellow, cyan, red

actin filaments

fix, dehydrate, coat with metal

fix, dehydrate, embed, section

• freeze rapidly, fracture, metal shadow
• freeze rapidly, image directly

• dry on support film in heavy metal solution
• dry, evaporate metal film at an angle
• freeze rapidly, image directly

• relevant for the question at hand
• genome available (not always necessary)
• ease of maintenance
• ease of manipulation
• size
• ease of analysis (transparency or to find veins)
• generation time
• cost and safety

• easy to obtain (grows in guts of humans)
• grows well in lab on borth
• 20 min duplication time
• serves to unravel fundamental processes such as: DNA replication, DNA translation into proteins, membrane protein

simple eukaryotic, single cell, has mito, small gemoable, amendable to manipulation: genetics, unravel processes such as cell division, basic biochemistry, metabolism, membrane trafficking

multicellular, has cholorplasts, gorws fast indoors; produces 100s of offspring in weeks, genome is sequenced, understanding food production, crop improvement, ecology

• multicellular animal with organs
• has a gut
• has neurons
• develops into 959 cells exactly
• transparent
• excellent genetics
• 70% of genes similar to human genes: study of human diseases
• understanding programmed cell death, life span analysis, development, neuroscience, behavior and cell division

• multi-cellular animal
• has a gut
• brain/neurons
• vast number of diff cell types (cell differentiation)
• larval development (how do legs, wing, etc. end up in right places)
• genes are similar to human genes (study human diseases)

• vertebrate animal
• pigmentless-white mice has transparent skin
• excellent genetics

planarian flatform, b/c they have stem cells t/o their bodies

axolotl

echinoderms; highest of the invertebrates, develops like a vertebrate, biochemistry, live cell imaging, fertilization, cell division, early development, primitive immune system

b/c of our common descent and the conservation of metabolic and developmental pathways and genetic material over the course of evolution

make random mutations, identify strains with relevant phenotype, map gene, sequence, gene, identify gene product for functional analysis

• classical genetics
• genomics
• isolation of genes and cDNAs
• biochemical purification of proteins

develop functional assay, homogenize cells, fractionate until product is pure, obtain sequence, isolate cDNA or gene for functional analysis

amplify gene by PCR; use DNA probe to clone gene, use expression cloning (functional assay for gene product in bacteria or eukaryotic cell); express gene product in bacteria or eukaryote for functional analysis

sequence genome or collection of ESTs, identify genes of interest for functional analysis

• sequence gene or cDNA, translate protein sequence (DNA sequencing by chain termination with di-deoxynucleotides)
• sequence protein

• stoichiometry from denaturing gel electrophoresis
• native molecular weight (ultracentrifuge, gel filtration)

X-ray crystallography, NMR (if <30 kDa)

• biochemistry means
• anatomy
• physiology

reconstitution from purified components

localization at the site of action

• reduced concentration of active proteins (drugs, antibodies, knockout, RNAi, dominant neg mutant)
• increased concentration of active protein (overexpression)
• replace native protein with protein having altered properties (classical genetics, homologous recombination

• postulate the biochemical mechanism
• write out differential equations describing the pathway
• stimulate the system behavior using available molecular concentrations, rate and equilibrium constants
• compare stimulations with data
• identify defects in model
• experiment to determine if defects arise from flaws in model or experimental data
• refine model, predict new properties and test by experimentation

• lipid bilayer (diff types allow for domains in membrane)
• proteins with function (in, out and through, significant for signaling, interactions, transport, ion, channels, structure, scaffold for carbohydrates)
• carbohydrates (interactions and signaling)

• phosphoglycerides-phospholipids
• shingolipids and glycosphingolipids 
• sterolds-cholesterol

• phophatidyl-choline, ehtanolamine, serine, inositol; differences in different membranes and in domains
• amphipathic, polar head, nonpolar tail
• contain choline, phosphate and glycerol with a double bond
• CDP to CMP

• derived from sphingosine, amino alcohol with long hydrocarbon chain and long chain fatty acid attached to sphinogosine
• glycolipids, gangliosides, sphingomyelin

• asymmetrically distributed
• amphipathic b/c of free OH

GS face outside; PS and PI face inside

cholesterol and sphingolipid

• cholestrol makes up about half the lipid in plasma membranes, but is much reduced or absent in intracellular membranes
• inner mito membrane is protein rich due to high density of respiratory chain enzymes

transporters, anchors, receptors, enzymes

transmembrane (integral protein), monolayer associated, lipid-linked, protein attached

hydropathy plots

peptide backbone is

hydrophobic effect

single alpha hellix about 20 residues @1.5 angstrom per residue

stabilizes the cytoplasmic surface of red blood cell plasma membranes

inherited deficiencies of membrane skeleton

around 600

hydrophobic