Lecture 27 Flashcards
How can defined changes in DNA sequence be made?
A mutant can be made through using a complementary strand to the gene you want changed with only a few bases changed in sequence (in some cases the original rather than new base will be changed)
An insertion can be made through using a strand slightly longer than the one on the vector
Conversely a deletion can be produced by a slightly shorter strand
Why do some pUC vectors have a bacteriophage derived origin of replication?
Allows for propagation of the molecule as a single strand
How are new genes synthesized in vivo?
The desired amino acid sequence is determined, this is then translated into the desired DNA sequence, then a series of overlapping oligonucleotides (100-150 bp) are synthesized these are annealed together and ligated to form the gene, these are then cloned and he clone is sequenced for confirmation
How do we ensure we get good yield from recombinant organisms which produce high value proteins?
Use a high level promoter with inducible expression, to optimise translation of the mRNA use codons which are commonly found in the host organism, to optimize stability use mutants that do not produce proteases, optimise the solubility and use humanized yeast if post translational modifications like glycosylation patterns are significant
How is DNA introduced to yeast?
Li+ treatment or electroporation
How is DNA introduced into Animal cells?
Ca precipitate of DNA
LIpid encapsulation
Microinjection
How is DNA introduced into plant cells?
polyethylene glycol, electroporation, biolistics and agrobacterium tumefaciens
What are the different Fates of DNA introduced into a eukaryotic cell?
Most DNA will make it into the nucleus, if an origin of replication is not present then very little expression will be seen and in most cases the nuclear material will be lost
Occasionally it will integrate into the host genome to form a stable transformant this can be targeted or random
If an origin of replication is present on the inserted DNA then integration and the formation of a stable transformant is much more likely
What are the selectable markers for eukaryotic cells?
Geneticin must be used for animals and yeasts, this could also be used for plants but typically kanamycin is used due to costs
How is kanamycin resistance expression induced in eukaryotic cells?
Use of an E.Coli gene Tn903 which inactivates all aminoglycoside antibiotics through phosphorylation
Its expression is done by a promoter taken from a double stranded DNA virus such as SV40 or CaMV 35S
These are viruses which have a wide range of hosts and allow for constitutive expression of the resistance gene
What are the characteristics of a reporter gene?
A gene used to measure the level of protein synthesis in a cell (not a selectable marker) used for good assays of gene product but cannot be used if there is background interference (so lac cannot be used in plants)
What are the common reporter genes?
Lac (E.Coli derived beta galactosidase)
Gus (E.Coli derived beta glucuronidase)
Luc (Firefly derived luciferase)
gfrp (jellyfish derived green fluorescent protein )
Why is yeast a good organism for gene transfer?
The complete sequence is known
Has eukaryotic post translational modifications
Has vectors for integration, high and low copy plasmids
What is the difference between gene transfer into yeast and higher eukaryotes?
Higher eukaryotes lack plasmids so integration must occur into the chromosomal DNA
What are the two kinds of DNA integration events?
Random which can occur anywhere on the DNA chromosome allowing only for gene addition and requiring dominant genes for phenotypes to be seen
Targeted which occurs via DNA homology, allows gene addition and replacement both dominant and recessive genes can be used
Why are targeted DNA integrations more powerful?
It allows the normal gene of the organism to be knocked out, creating mutants useful for genetics studies
