Techniques to Study Gene Regulation Flashcards
What is a cis-regulatory sequence?
Regulation done by the gene and sequences in the gene
What is trans-acting factor?
Regulation done by something else other than the gene itself, like a protein binding
What are we trying to figure out when studying gene expression?
What sequences are regulating gene expression and what proteins bind to those sequences
What techniques will look at the levels or RNA or proteins in a cell?
Western blot, Northern blot, in situ hybridization, RT-PCR, q-RT-PCR, microarray, immunofluorescence
What is the conceptually simplest way to determine if a sequence is regulating gene expression?
Mutate it and see what happens
Why is it difficult to mutate a suspected regulatory sequence to see what happens?
Hard to make a targeted and specific change in the genome. The gene may also be essential and it is difficult to study gene expression when the organism is dead
Why do we use reporter genes?
Their expression is easy to detect and eliminates the need to design primers, probes, and antibodies for the other gene
How are reporter genes used to determine if a sequence of interest is regulatory?
Put the sequence of interest upstream of a reporter gene and detect how expression of the reporter gene changes
What is promotor mapping?
Putting a sequence of interest in front of a reporter gene and cutting out bits of that sequence to determine which parts of the sequence are important
What is enhancer identification?
Placing a sequence of interest very far upstream of a reporter gene to determine if it is acting as an enhancer
What is an EMSA?
Electromobility shift assay. Determines if a protein is binding to DNA by looking for a shift created by the bound protein in the gel
What does an EMSA allow us to answer?
Does this protein bind to a specific sequence of DNA
What creates the shift seen on an EMSA gel?
Protein bound to DNA. It is much larger and will run a lot slower than the unbound DNA
What is labelled in an EMSA?
The DNA
What is DNase footprinting?
Using DNase to cut a labelled DNA sequence to determine where a protein is binding. DNase can’t cut any DNA bound to a protein, so when the fragments are run out on a gel there is a gap - the footprint - where the protein was bound
What does DNase footprinting allow us to answer?
What is the specific DNA sequence that a protein binds to
What is labelled in a DNase footprinting assay?
One end of the DNA
What is chromatin immunoprecipitation?
Breaking up the chromatin so that the histones stay bound and using immunoprecipitation to precipitate out the protein of interest bound to DNA. Then separating the proteins from the DNA to determine what the sequence is
What does chromatin immunoprecipitation allow us to answer?
What are all the sequences in a cell that a protein of interest binds to
What is RNA interference?
A regulatory mechanism that occurs in eukaryotic cells at the level of mRNA and translation
What is miRNA?
A short piece of RNA that complementary base pairs to mRNA
How is miRNA produced?
It is transcribed into a long transcript and processed into a 70 nucleotide hairpin by the microprocessor complex in the nucleus. Then exported into the cytoplasm and is processed into a 21 nucleotide duplex by dicer. It is bound by the RISC complex which selects the strand of miRNA that is complementary to the mRNA
What is responsible for processing the long transcript of miRNA into a 70 nucleotide hairpin?
Microprocessor complex
What is responsible for processing the 70 nucleotide hairpin into 21 nucleotide duplexes of miRNA?
Dicer endonucleases
What is responsible for selecting the correct strand of miRNA that is complementary to the mRNA?
RISC complex
What happens to the mRNA when miRNA is perfectly complementary?
It gets rapidly degraded
What happens to the mRNA when miRNA has imperfect complementarity?
Translation is blocked
Are any miRNAs activators?
No, they only decrease gene expression
How common is gene regulation by RNA interference?
Very common, over 50% of human genes are regulated by RNA interference
How do researchers use RNAi as a technique?
By introducing double stranded RNA into the cell, where dicer enzymes recognize that and cut it into siRNA with the same function and is complementary to the mRNA we want to knock down expression
Why do we use RNAi as a technique?
It is a great way to shut off gene expression to see what a gene does, and is easier than making mutations
What does RNAi allow us to answer?
- What does this gene do?
2. Where is this gene important
