Samenvatting van de hoofstukken 8, 15, 17, 18 en 20 van Molecular Biology of the Cell by Alberts et al. Merendeels engels. Hoofdstukken zijn nodig om te leren voor de toets Moleculaire Celbiologie (toen 8RB00) op de TU/e. Gemaakt voor 2e jaars en geschreven
Isolating individual cells: disrupt extracellular matrix. Maak cellen los. Enzymes
digest extracellular matrix. Labeled antibody that binds to one specific cell type
is brought into the substance. Separation by electronic fluorescence activated
cell sorter. The fluorescent cells are separated by electrical charge.
Also a selected piece of tissue can be cut out by a laser beam.
Cells can only divided for a number of times: to fix this you need to highly
express recombinant telomerase. And you need to surpress the check-point
mechanisms that eventually make a cell stop dividing. This last one can be done
by introducing cancer oncogenes.
Stam cellen kunnen oneindig prolifereren en differentieren in alle cellen. Onder
goede condities zullen stamcellen uitgroeien naar je gewenste cel.
Hybridomas: cellen die oneindig antibodies kunnen maken.
Analyzing and manipulating DNA:
Restriction endonucleases: cut DNA at specific sites double strands. Cleaves DNA
into fragments of strictly defined sizes. These sequences in own DNA are
methylated so that bacteria don’t cut own DNA. Restriction nucleases are derived
from bacteria. Palindromic.
Northern blotting: selectinf mRNA on size. Gel electrophoresis adding labeled
DNA probes.
Southern blotting uses DNA instead of RNA. DNA probes will hybridize with
complementary strands and labeled will be visible by autoradiography.
Cloning: inserting a DNA fragment into a DNA genome that is of a self-replicating
virus or plasmid. Plasmid vector: small circular molecules of double stranded
DNA. Purified plasmid is cut by restriction nucleases linear DNA The to be
cloned DNA is cut by same nuclease added to plasmid circle reformed
brought into bacteria with antibiotic region cloning. Plasmid used to make DNA
libraries.
cDNA: DNA that is made by using RNA template. Done by reversed transcriptase.
cDNA doesn’t have introns. Uninterrupted coding sequence.
PCR: good results after few rounds. Chain reaction. Heat proof polymerase is
needed. Primers needed that are complementary with the selected DNA region.
Celbiologie hfdst 15
Unicellular organisms can influence each other’s behavior. Respond to chemical
signals secreted by neighbors: quorum sensing.
Multicellular animals communicate by signal molecules. These molecules can be
in the extracellular space by exocytosis, on the cells surface or emitted through
diffusion. Target cell uses receptor to respond. Receptor is transmembrane.
, Paracrine should not diffuse to far, enzymes will clean them up and rapid
diffusion to target cell. Response change is different per target cell. Alternation in
gene expression takes longer than phospolyration of already present proteins.
Gap junctions for intercellular signaling.
Cells can grow +divide, differentiate or survive in presence of signal molecules.
But the effect is mostly dependent on the concentration of the signaling
molecule. No signals can lead to dead. A combination of receptors, signals and
pathways is required. Signal molecule can have different reactions to different
celltypes. Actyl choline lowers contraction of heart muscle but stimulates
contraction in skeletal muscle. Even if signal and receptor are the same, different
cells can have different pathways and therefore different outcomes.
Intracellular receptors. Signal molecules involve NO and steroid hormones. These
are hydrophobic and small to pass the plasma membrane. NO (gas) relaxes
smooth muscle in blood vessel. Neuron acth chl in synaps target cell
activates NO synthase NOS NO production diffusion to smooth muscle
binds NO guanylyl cylase cyclic GMP relaxation.
Intracellular can be for gene expression. Steroid hormones. Alter ability of
receptor proteins to control transcription. So receptor is also effector protein.
Bind specific DNA sequence. Before activation, bind to inhibitor proteins and can
be in nucleus bound to DNA or in cytoplasm.
Cell surface receptor: ion gated. Ligand bind, receptor gate opens, ion can enter.
GPCR activates membrane bound target.
Enzym coupled act as enzyme.
Surface receptors act as a switch: turned on and off.
1. Dephospholyration by protein phosphatases or phosphorylation by kinases.
2. GTP binding proteins.
Induced proximity: signal triggers assembly of signaling complex.
Cells can adjust their sensitivity to signals. Cell will respond to changes in signal
concentration instead of to the absolute concentration. Receptors can be
destructed or removed for a while. Inactivating receptors or signaling molecules.
GPCR: after binding, activates G protein. G protein bbinds receptor after
activation. 3 subunits. Alfa binds GDP/GTP. After GPCR activation, it swaps GDP
for GTP in alfa. Valt uiteen tot alfa en betagamma.
Alfa part activates adenylyl kinase who makes cAMP. cAMP activated PKA.
Inactive PKA has a regulatory unit and an inactive catalytic subunit. cAMP binds
regulatory unit and frees the other unit.
G protein activates PLC PIP2 to IP3 and DAG. IP3 opens ca2+ release channels
in ER. DAG activates PKC. PKC uses ca2+ . 4 Ca bind calmodulin voor extra
activeringen.
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