1: L2-3 Flashcards
Define genome editing and its requirements
forces changes to the sequence of DNA in a controllable manner. In living cells, efficiently and permanently
2 Historical version of Genome editing and their problems
- Selective breeding
- Relies on 1. Natural variants or 2. Random mutations - Designer DNA binding proteins
- Problem: A new protein needs to be designed and made for each new DNA target site = $$ and time
What does CRISPR stand for
Clustered Regularly Interspaced Short Palindromic Repeats
How does CRISPR work
a. Cas9 is paired with an artificially created short guide RNA (gRNA, a molecular strand complimentary to a precise, disease-causing mutated section of DNA)
b. gRNA guides Cas9 to its corresponding section of the genome and induces a break in both DNA strands (DSBs), a ‘cut’.
Advantages of CRISPR
Multi-cut= multi gene editing
easier than making 3d protein
How does DNA repair itself in CRISPR
- Non homologous end joining
2. homologous recom
How does homologous recombination work
sister chromatin used as template for missing DNA sequence - CAS9 + rna + template sent
3 applications of CRISPR
- Add new sequence= homologous
- modify existing sequence (point mutation example)= homologous
- delete DNA = non homologous
what’s a gene drive
o Used for population control of pests
- introduces suicide gene that’s passed on or one that eradicates fertility
Limits of CRISPR
‘off target’ cutting
PAM site limits the choice of DNA sites to be cut
Cas9= large protein (hard to fit in virus)
how to harness the repair mechanism of cell -carry out genome editing
-
When adding new seuqence to DNA
= Harness the ‘homologous recombination’ mode of DNA repair
- Break at point of new sequence, artificially introduce small piece of DNA used as template of repair
- Either side of DNA wanting to repair= high amount of homology (similarity)= trick to prompt central foreign DNA into genome
Describe the nuclear envelope
- double-membrane: inner and outer membrane
- outer membrane = continuous with Rough ER
- nuclear pores = allows entrance and exit of molecules
describe protein synthesis and transport
a. Gene prescribed onto mRNA in nucleus,
b. Translated into proteins in RER via nuclear pores
c. Moved to Golgi apparatus for sorting packaging and modifying
d. If protein to be moved outside of cell= transport vehicles or lysosomes
Describe the features and function of the RER
- Cisternae/lamellae (sheets)
- Studded with ribosomes (protein/RNA subunits)
- Site of Protein synthesis
Describe the features and function of the SER
- Tubular network
- Steroid + cholesterol synthesis
- Drug metabolism
What does the geologic apparatus do
• Sorts, modify or package the protein
how is the golgi similar/different to RER
- Similar to RER as both have sheet like structures
* Golgi Difference= bulging ends
What are lysosomes and what do they do
- Lysosome created by golgi
* Digests unwanted molecules and ‘Recycles’
3 ways molecules move across cell
diffusion
facilitated diffusion
active transport
describe diffusion
- Lipid- soluable solutes (can pass freely through phospholipid membrane
- E.g. steroid hormones, gases (O2, CO2)
- Passive transport (no extra energy) - driven by concentration gradient
describe facilitated diffusion
- Channel or carrier protein- mediated
- Too large or lipid insoluble i.e. glucose (uses glucose transporters)
- Passive transport (no extra energy) - driven by concentration gradient
explain saturability of facilitated diffusion
• Saturable: only x amount of carrier proteins // if high concentration outside cell, but only few carrier proteins // limited rate of entry + saturation
o Can increase carrier proteins
• i.e. insulin triggers uptake of glucose transporters in membrane in cells then internalised after uptake complete = regulation of function
describe active transport
- Carrier-mediated
- Energy required (from ATP)
- Enables transport AGAINST a concentration gradient
- eg: amino acids (builds proteins // needs large amount), ions (sodium-potassium pump)
- Three main types of Integral membrane proteins
(1) Channels: not change conformation when molecules pass through, open and shut, passive
(2) Carriers: change of conformation when molecule passes through, Active OR passive transport
(3) Receptors: required for CELL SIGNALLING
2 types of hormones and what they bind to on membrane (+ examples)
- Steroid hormones bind intracellular (inside cell) receptors Eg. Estrogen, testosterone, cortisol
- Peptide hormones bind membrane receptors, docked in membrane + are ligands Eg. Oxytocin, insulin, growth hormone
