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what does the receptors and signaling mechanisms allow cells to do?
adapt to environmental conditions
what does the ligand bind?
receptor to turn it on
what does receptor activate?
what happens after enzymes are activated by receptor?
make second messenger cAMP
what does cAMP activate?
what do kinases do?
phosphorylate and active enzymes
what is the job of feedback mechanisms?
to control molecular composition, growth and differentiation
what are some checkpoints in the cell cycle?
check for DNA nicks, check for favorable environmental conditions, check for damaged or unduplicated DNA, check for chromosome attachment to mitotic spindle
list the order of the three domains of life in the phylogenetic tree
archaea, bacteria, and eucarya
explain the prebiotic origin of life
- 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
what is divergence?
identical genes diverge from a common ancestor due to a mutation (sister lineages)
diverged as a result of mutations in sister lineages
what are orthologous genes?
proteins with same functions but found in different species
what are the two scenarios for the origin of eukaryotes?
- 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
speculation on the origin of organelles in eukaryotes
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
what are the functions of membrane?
compartmentalization, scaffold for reactions, selective permeability barrier, transport, signal transduction, intercelluar interactions, energy transduction.
how are membrane proteins positioned in the bilayer?
- 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
how can we test the mobility of membrane proteins?
- laser trap (immobile proteins do not diffuse freely and particles attached resist displacement
how are membrane proteins solubilized for biochemical studies?
using nonionic detergents
how to extract membrane proteins?
integral proteins can be extracted with nondenaturing detergents, peripheral proteins can be extracted using high salt (to disrupt protein-protein interactions
what is the role of peripheral membrane?
- scaffold for signal transduction
- skeletal support
- cell-cell communication and interaction
what is FRAP (Fluorescence recovery after photobleaching) used to measure?
lateral diffusion rates in membrane
what are the components of reductionist strategy?
- def of ?
- inventory of parts
- concentration (biochm, microscopy)
- partners (biochm, genetics)
- rate and equilibrium constants
- biochemical reconsitition
- mathematical model
- physiological tests
what are some cells that you can see without microscopes?
- Xenopus egg
- most other cells
what is the difference between TEM and bright field?
- You don't have to kill the cell in TEM, but you do in brightfield
- light microscope is limited by point spread function
what is deconvolution?
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
fluorescent dye bound to protein
needs to be microinjected
fluorescent dye bound to lipid need to
be injected or fused to cell
fluorescent dye bound to nucleic acid
in situ hybridization in fixed cells
fluorescent dye bound to ligand for cellular molecule
antibody isolated from animals immunized with target molecule
where does GFP come from (green fluorescent protein)
- cDNA from jellyfish can be fused to genes for many proteins and expressed in live cells
- variants are yellow, cyan, red
what does the fluorescent phalloidin bind?
how to prepare cells from scanning EM?
fix, dehydrate, coat with metal
how to prepare cells for thin sections on TEM?
fix, dehydrate, embed, section
how to prepare cells for freeze-fracture and frozen hydrated?
- freeze rapidly, fracture, metal shadow
- freeze rapidly, image directly
how to prepare molecules in neg stain, shadowing, frozen hydrated?
- dry on support film in heavy metal solution
- dry, evaporate metal film at an angle
- freeze rapidly, image directly
what makes a good model organism?
- relevant for the question at hand
- genome available (not always necessary)
- ease of maintenance
- ease of manipulation
- ease of analysis (transparency or to find veins)
- generation time
- cost and safety
why is E. Coli an excellent model organism?
- 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
what is great about brewer's yeast?
simple eukaryotic, single cell, has mito, small gemoable, amendable to manipulation: genetics, unravel processes such as cell division, basic biochemistry, metabolism, membrane trafficking
why is arabidopsis thaliana a model organism?
multicellular, has cholorplasts, gorws fast indoors; produces 100s of offspring in weeks, genome is sequenced, understanding food production, crop improvement, ecology
why is nematod worm Caenorhabditis elegans a good model organism?
- multicellular animal with organs
- has a gut
- has neurons
- develops into 959 cells exactly
- 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
why is drosophila melanogaster a good model organism?
- multi-cellular animal
- has a gut
- 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)
why s the mouse Mus musculus a model organism?
- vertebrate animal
- pigmentless-white mice has transparent skin
- excellent genetics
what can regenerate the whole body? why?
planarian flatform, b/c they have stem cells t/o their bodies
what organism can regenerate its limbs?
what are some historic non-genetic model organisms?
echinoderms; highest of the invertebrates, develops like a vertebrate, biochemistry, live cell imaging, fertilization, cell division, early development, primitive immune system
how is studying model organisms relevant from one species to another?
b/c of our common descent and the conservation of metabolic and developmental pathways and genetic material over the course of evolution
what is classical genetics?
make random mutations, identify strains with relevant phenotype, map gene, sequence, gene, identify gene product for functional analysis
what are the molecular inventories?
- classical genetics
- isolation of genes and cDNAs
- biochemical purification of proteins
what is the biochemical purification of proteins?
develop functional assay, homogenize cells, fractionate until product is pure, obtain sequence, isolate cDNA or gene for functional analysis
how do we isolate genes and cDNAs
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
how do we do genomics?
sequence genome or collection of ESTs, identify genes of interest for functional analysis
how do we determine the primary structure of DNA?
- sequence gene or cDNA, translate protein sequence (DNA sequencing by chain termination with di-deoxynucleotides)
- sequence protein
how do we determine the subunit composition of DNA?
- stoichiometry from denaturing gel electrophoresis
- native molecular weight (ultracentrifuge, gel filtration)
how do we determine the atomic structure of DNA?
X-ray crystallography, NMR (if <30 kDa)
what are some tests of physiological function?
- biochemistry means
what are the biochemistry tests of physiological function?
reconstitution from purified components
what are the anatomic tests of physiological function?
localization at the site of action
What are the physiological tests of physiological function?
- 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
describe the process in coming up with mathematical models
- 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
what is the composition of the plasma membrane
- 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)
what are the different types of lipids?
- shingolipids and glycosphingolipids
what are phosphoglycerides?
- 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
what are the shingolipids?
- derived from sphingosine, amino alcohol with long hydrocarbon chain and long chain fatty acid attached to sphinogosine
- glycolipids, gangliosides, sphingomyelin
what is special about the sterols
- asymmetrically distributed
- amphipathic b/c of free OH
which direction does GS face in the biological membrane?PS and PI?
GS face outside; PS and PI face inside
what are rafts rich in?
cholesterol and sphingolipid
what is the difference between inner membrane and plasma membrane
- 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
how are membrane proteins classified?
transporters, anchors, receptors, enzymes
what are some ways proteins associate with membrane?
transmembrane (integral protein), monolayer associated, lipid-linked, protein attached
what are the plots that monitor the hydrophobicity of the integral membranes?
what part of the membrane protein is hydrophilic?
peptide backbone is
what effect underlies the disposition of integral membrane proteins in the bilayer?
what is the shortest way to span a membrane?
single alpha hellix about 20 residues @1.5 angstrom per residue
what is the purpose of a membrane skeleton?
stabilizes the cytoplasmic surface of red blood cell plasma membranes
what causes fragile red blood cells and hemolytic anemia?
inherited deficiencies of membrane skeleton
channel has low selectivity for molecules with what molecular weight?
what kind of side chains do amino acids have?