Q/A Session 4/5 Flashcards
(30 cards)
What is a receptor?
Protein molecule that receives chemical signals from outside the cell
What is a ligand?
Molecule/drug that binds to a receptor
When alcohol binds to the GABAa receptor what enters the cell?
Cl- ions into the cell
What is the role of HIV protease?
Cleave long proteins into fragements
What kind of inhibitor will a pharma drug be?
A competitive inhibitor
What do transcriptional factors do?
Determines when and in what cell a gene is transcribed to produce mRNA
What are transcription factors?
Proteins that bind to a specific DNA sequence and control rate of transcription
What is a promoter?
DNA sequence at which transcription factors bind and recruit RNA polymerase
What does a promoter determine?
If and how much a gene is transcribed
What 4 things does PCR require?
- DNA from cell
- DNA from nucleotides
- Primers with sequence that can base pair to region of amplification
- Taq DNA polymerase
What are the 3 steps of PCR
- Heat DNA to 95 degrees to separate DNA strands. Heat breaks the H-bond
- Cool to ~60 degrees to base pair a DNA primer. Primer=short DNA sequence of nucleotides complementary to strand
- Heat DNA to 72 degrees to allow Taq DNA polymerase to copy DNA.
What do tumour suppressor genes do?
Encode proteins that prevent uncontrolled cell growth
What do onco-genes do?
Promote cell division
What is a proto-oncogene?
A normal gene that when mutated promotes uncontrolled cell growth
What are the components of recomb DNA plasmids?
- Origin of Replication
- Allows initiation of replication using host DNA polymerase - Antibiotic Resistance gene
- Allows selection of cells containing plasmid - Promoter
- Drives expression of gene in cells with appropriate transcription factor machinery
What are the characteristics of plasmids?
- Circular pieces of dsDNA
- Replicate independently of host chromosomal DNA, but use host machinery
- Provide a benefit to host
What are restriction enzymes?
- Cut dsDNA into specific sequences creating sticky ends
2. Found naturally in bacteria-degrade foreign DNA
What is transformation?
Transfer of plasmids into bacteria
What are the 5 steps of producing recombinant protein in prokaryotes?
- Isolate gene of interest
- Clone into expression plasmid
- Transform bacteria for expression
- Grow cells expressing gene of interest
- Isolate and purify protein
Advantages of prokaryote systems
- Low cost
- high yield
- pathogen free
Disadvantages of prokaryote systems
- Proteins often partially folded
- Inability to perform post-translational modifications
Advantages of mammalian cell recombs
- Protein can be produced as pre-pro-protein and processed efficiently
- Secreted from cells: easier purification
Disadvantages of mammillian systems
More expensive
What are the 5 steps of producing recombinant protein in eukaryotes?
- Isolate cDNA
- Clone into eukaryotic expression plasmid
- Transform bacteria to produce more plasmid DNA and then transfect eukaryotic cells
- Extract recomb from cell media.
- Purify
