Bio Test 3 Flashcards
Southern Blot
DNA is usualy digested with restriction enzymes first. Then, DNA is transferred to a filter for hybridization. Electrophoresis is generally on agarose gel.
Northern blot
RNA is on the filter. Agarose gels, usually. Probe as for southern.
Western
Protein is on the filter. SDS Polyacrylamide gels. Probe is an antibody to the protein of interest
Probe
Labeled nucleic acid complementary to the sequence that we are looking for. Electrophoresis is usually on agarose gel.
Why do we need a blot
to see a particular part of the genome, or a particular RNA of protein.
Blotting involves…
transfer of the DNA to a nylon or nitrocellulose membrane.
Probing involves…
soaking the filter in a solution containing a labeled DNA that is complementary to the gene region that we want to observe
What are antibodies
glycoproteins produced by our B lymphocytes, they recognize foreign antigens and are crucial to the immune system
what do we produce antibodies against
bacteria, viruses, etc
cytoskeleton
cell shape and movement
ex of Organelles
Nucleus mitochondria Chloroplasts Peroxisomes Secratory System Endocytic system
Secratory system includes…
ER, Golgi, Transport vesicles, endosomes, lysosomes, plasma membrane
endocytic system includes…
lysosomes and endosomes
cytosol
contains many metabolic pathways. Protein synthesis
Nucleus
contains main genome; DNA and RNA synthesis
Endoplasmic Reticulum
synthesis of most lipids; synthesis of proteins for distribution to many organelles to another organelle
Lysosome
intracellular degradation
Golgi apparatus
modification, sorting, and packaging of proteins and lipids for either secretion or delivery to another organelle
endosome
sorting of endocytosed material
mitochondria
ATP synthesis by oxidative phosphorylation
Chloroplasts
ATP synthesis and carbon fixation by photosynthesis
peroxisomes
oxidation of toxic molecules
Proteins that need to go anywhere in the secratory or endocytic system are synthesized on what?
Membrane-bound ribosomes
nucleus structure
pore protein and lamins
nucleus proteins coming and going
importins and exportins
mechanisms for coming and going of proteins
importins, exportins and the GTP binding protein, RAN.
Nuclear envelope
two lipid bilayers contiguous with the ER
Nuclear lamina
fiborous network of proteins underneath membrane and reaching throughout
nuclear pores
complicated protein structures to let molecules in and out of the nucleus
Lamins
type of intermediate filamnt present in nucleus inside nuclear membrane
nucleoporins
proteins of the nuclear pore
karyopherins
importins and exportins for moving proteins and RNAs in and out of the nucleus
G-Proteins
Ran is an example of an important type of molecular “switch” involved in many cellular processes
what is the role of the nuclear envelope?
seperate the nucleus from the cytoplam
What two membranes does the nuclear envelope consist of?
nuclear pore complexes and nuclear lamina
the outer membrane is continuous with what system?
ER
the inner membrane includes proteins that bind to what?
Nuclear lamina
most molecules that go in and out need to go through what?
nuclear pore
How do larger molecules get in and out of the nucleus
they need to imported and exported
importins
bind the protein to be imported by means of the nuclear localization signal and then brings it to the nuclear pore and then through it.
exportins
bind to proteins to be exported from the nucleus using the nuclear export signal located in these proteins. (NLS)
RAN
example of a GTP binding protein that is involved in many cellular processes.
Ran binds to GTP and allows cargo to be released. escort the imp out to get another cargo to put it into the nucleus.
Signal recognition particle
the complex moves to the translocation channel, where translation of the protein occurs while it is being threaded across the membrane.
signal peptidase
remove the signal sequence
anterograde trasnport
forward in the ER to Golgi to vesicles to plasma membrane.
retrograde transport
movment of substance in the reverse of anterograde. Golgi back to ER
Coated vesicles
COP for vesicles going from ER to Golgi and back
Clatherine for going to plasma membrane, lysosome, secratory vesicles and in the reverse direction
gathering of proteins to be put into vesicles for transport
Cargo receptor cargo, with signal to recognize receptor adaptin, specialized for particular receptors clatherine, coting vesicles dynamin, to pinch the vesicle off
what releases the vesicle at the neck between the donor membrane and the vesicle
dynamin
v-SNARE’s
for membrane fusion
t-SNARE’s
interact with v-SNARE’s
Rab proteins
small GTP-binding proteins, similar to the nucler Ran proteins. Give each vesicle type its own identity, marking it for where it should go
Describe docking at the target membrane
Rab is bound by tethering proteins on the surface. Once there v-SNARE’s and t-SNARE’s can interact, forming coiled-coil protein complexes that bring the membrane into contact.
membrane fusion
proteins and lipids in the vesicle become part of the target membrane. Soluable material inside the vesicle goes into the new compartment or the outside of the cell.
what happens when vesicles arrive at the target membrane
vesicles actually move into the cell by means of motor proteins that walk along actin and microtuble filaments.
arrive and fuse
proteins recognize the correct membrane so fusion can be done correctly
ER
addition of sugars to asparagine residues in proteins
formation of disulfide bonds
protein quality control, destruction of wrongly folded proteins
what are the flat-looking membranous structures in the stack of membranes
cisternae
trans golgi network
major branch point for determining ultimate destination: constitutive secretion regulated secretion, movement to lysosomes via late endosomes.
Mannose-6-phosphate
a signal that the protein should go to the lysosome eventually
constitutive secretion
for things like normal membrane proteins and lipids of this cell type. Replacements are being made for regular
membrane molecules.
regulated secretion
Molecules are held in vesicles
until a signal causes them to move to the surface for release. Example: neurotransmitters
Endosomal/lysosomal pathway
Enzymes like acid hydrolysases normally are found in the lysosome. They are sorted in the TGN to the lysosomal compartment.
endocytic system
movement of materials into the cell shares some compartment with the secratory system.
Endosomes and Lysosomes
Actin
the filament that forms all of these networks, branches, crosslinks.
SRP
starts with a recognition particle. Docked at the ER with srp, so the protein can get to their destination.
Endocytosis
use clatherin to make the little basket-like structures that form the vesicles.
Going INTO the cell
Step 1: endosome.
Endosomes
the sorting compartments.
early endosomes
just beneath PM
late endosome
closer to the nucleus
LDL receptor
take up cholesterol-containing particles from the blood.
If the receptor is not functioning normally cholesterol accumulates to very high levels in the bloodstream and forms deposits in the arteries, skin and tendons
Receptor-mediated endocytosis, LDL example.
low-density lipoprotein.
receptors exist on the surface of some cells
Binding of LDL to receptor triggers formation of clathrin-coated vesicles, vesicles fuse with the endosome, the endosome matures to for lysosome, cholesterol is released into cytosol.
Cystic Fibrosis
results from probles getting the CF protein to the cell membrane.
Intermediate filaments
structural support
keratins, filamentous proteins of skin, hair, nails.
microtubules
chromosomes movement and movement to vesicles and for structure and movement of cilia and flagellae.
-Hollow tubes of proteins that are comprised of alpha and beta tubulin dimers.
actin filaments
most generally associated with cell movement, cytokinesis, shape changes.
What does Coiled-coil mean
that they wrap around each other to form strong filaments.
Dynamic instability of microtubules
GTP-bound form is added at the plus end
GTP is eventually hydrolyed
GDP-bound form is less stable
Dynamic instability means what?
individual microtibles shrink very rapidly and then grow back
How do you stabilize microtubules?
by anchoring them to capping proteins in the cell membrane.
Actin polymerization
Forms thin filaments
Plus and minus end
they are NOT hollow the way microtubules are but they can form different types of networks.
What are forms of actin filaments
linear
branched
networks
bundles
where is cortical actin found
just beneath the plasma membrane
here are stress fibers found
bundles of actin associated with cell movement
what do actin bundles form?
they form the structure of “microvilli”
where do networks usually occur?
just underneath the plasma membrane
three types of motor proteins?
Myosins
Kinesins
Dyneins
Myosins
movement along actin filaments in the plus direction
Kinesins
movement along microtubules in the plus direction
Dyneins
movement along microtubules in the minus direction
Microtubules organizing centers
centrosome is the main one
centrosomes
near nucleus
organizes microtubules
minus ends are anchored here by the gamma-tubulin complex
plus end go out into the cell
Centrioles
resemble the MTOC’s at basal bodies.
when present, they exist in pairs and are comprised of tubulin
cytoskeleton motor protins
“head” is the microtubule-binding portion that includes ATPase to provide energy
“Tail” is everything else: cargo binding, dimerazation, regulation of motor activity.
Why are the tail ends different from each other in a motor protein?
they bind different cargo, different localization in the cell, and different regulation
describe one ATPase cycle
ATP binding
hydrolysis
release of ADP and phosphate
transport of microtubules in axons
plus end of microtubule is in the far end of the axon, minus end is in the cell body.
Synaptic vesicles
proteins synthesized in cell body need to go all the way to the end of the axon! They use KINESINS for this purpose
anterograde …go toward plus end.
What does ATP carry
phosphate in a high energy linkage
what does NADH carry?
electrons and hydrogens for “reducing power”
what does Acetyl co-A carry
acetyl groups
Glycolosis in cytosol
glucose is converted to pyruvate.
we gain 2 ATP and 2 NADH per glucose. Pyruvate is formed and goes to the mit.
Pyruvate is oxidized to acetyl coA forming one NADH and releasing one CO2. Goes to TCA cycle.
TCA cycle
Krebs cycle: acetyl coA from pyruvate is oxidized to CO2, generates one NADH at the beginning.
the reduced forms of NAD and FAD are formed (plus GTP)
what is the flow of protons back into the matrix called?
ATPsynthase.
what does ATPsynthase lead to
ATP production
What does the transfer of electrons from NADH through electron transport chain lead to?
A Protein gradient across inner mit membrane
Cristae
the folds in the inner membrane of the mitochondrial membrane. This is where electron transport happens. ATP synthase is also in this membrane.
inner membrane space
protons are pumped to this region forming the gradient that is used for ATP production
Pyruvate dehydrogenase complex
oxidizes pyruvate to acetyl coA and CO2, CO2 is released.
reaction produces NADH
acetyl coA is the molecule that enters the TCA cycle
Fatty acid oxidation
lipids and fatty acids are broken down into two-carbon units and enter the TCA cycle as acetyl coA
Uses carbon and energy from sugars to produce ATP. Liberates CO2 to the atmosphere from the carbon compound that it is using. Uses O2 as the final electron acceptor following oxidation of sugars.
Mitichondria
light reactions
harvest energy from light to create ATP and NADPH by means of high energy electrons for reduction
-thylakoid membrane
dark reactions
for capturing CO2 from the atmosphere to create the complex carbon compounds that we need to live
-occur in the stroma
chlorophyll
light harvesting compound presents in reaction centers that absorbs light
photosystem II
creates a proton gradient for formation of ATP by ATP synthase.
b6f complex
this complex pumps protons, contributing to the proton gradient that eventually forms ATP through ATP synthase
three phase of the calvin cycle
carbon fixation
reduction
regeneration of intermediates
carbon fixation
CO2 is combined with a five-carbon bi-phosphorylated sugar called 1,5 ribulosebisphosphate, forming an unstable 6-carbon compound.
Enzyme is called “rubisco”
reduction
Use NADPH and ATP to make glyceraldehyde 3 phosphate. this is used to make glucose in a series of steps that are not part of the calvin cycle
Glyceraldehyde 3 phosphate
is a triose phosphate and is one of the intermediates in glycolysis
1 is produced for every three turns of the cycle
regeneration
regenerate the 5 carbon compound ribulose bisphosphate that can accept another CO2.
