Study Flashcards
Manipulations of Recombinant DNA
Cleavage of DNA at specific sites by restriction nucleases which greatly facilitates isolation and manipulation of individual genes
DNA ligation which makes it possible to design and construct DNA molecules that are found in nature
DNA cloning through the use of cloning vectors or the polymerase chain reaction in which a portion of DNA is repeatedly copied to generate many billions of identical molecules
Nucleic acid hybridization which makes it possible to find specific sequence of DNA a=or RNA with great accuracy and sensitivity on the basis of its ability to selectively bind a complementary nucleic acid sequence
Rapid determination of the sequence of nucleotides of any DNA making it possible to identify genes and to deduce the amino acid sequence of the proteins
Simultaneous monitoring of the level of mRNA produced by every gene in a cell using nucleic acid microarrays in which tens of thousands of hybridization reactions take place simultaneously
Morphological changes in apoptosis
cleavage of chromosomal DNA by endonucleases
• loss of the electric potential across the inner mitochondrial membrane
• movement of phosphatidylserine to the outer portion of the phospholipid membrane, which signals
the macrophages to phagocytise the apoptotic bodies without initiating an inflammatory response
Intrinsic factor
process of cell death triggered by the internal signals
happens in the mitochondria
healthy cells have a protein called Bcl-2 that inhibits apoptosis
When cell is damaged Bax ( protein) is stimulated and attaches next to Bcl2 to prevent Bcl2 from inhibiting apoptosis. Bax also punctures holes in the outer membrane leading to the release of cytochrome c into the cytoplasm. Cytochrome c causes aggregation of a protein complex apoptosome which binds to and activates a group of protease called caspase 9
Caspase 9 proteins cleave and break down the DNA and other internal structure with the cell that eventually kill off the cell
Extrinsic factor
signal molecules originating outside the cell stimulate apoptosis
Healthy cells contain special integral ,membrane proteins known as death receptors that can bind complementary molecules called death activators. It stimulates an internal process that activates caspase 8 proteases. Their proteins then activate a cascade that leads to the destruction of the cell
Apoptosis inducing factor
does not use caspases but AIF
AIF is located in the intermembrane space of the mitochondria. When the cell commits to apoptosis AIF is released into the cytoplasm and eventually makes its way into the nucleus where it binds to the DNA and triggers destruction of DNA
this type of mechanism is controlled by the neurons
Proteins targeted by caspases
nuclear laminins – result in the breakdown of the nuclear membrane
• exonuclease inhibitors – this allows the specific exonucleases to become active and cleave the DNA
in the nucleus
• cytoskeletal components – cells round up
• cell-cell adhesion proteins – cells detach from their neighbours
Western blotting
analytical technique used to detect specific proteins in a mixture of proteins. It is often used as a follow up test to confirm the presence of an antibody and help diagnose a condition. Protein sample is prepared by mixing it with SDS detergent which makes proteins unfold into linear chains and coats them with negative charge. protein molecules are separated according to size using gel electrophoresis. Protein are separated from gel onto a blotting membrane. The membrane carries all the protein bands originally on the gel
Agarose gel electrophoresis
separating large fragments of DNA/RNA from one another by size
3 mechanisms for replication
replication is the process of making copies of DNA
Initiation:
Elongation:
Termination:
