Techniques to Study Gene Regulation

Question 1

What is a cis-regulatory sequence?

Answer 1

Regulation done by the gene and sequences in the gene

Question 2

What is trans-acting factor?

Answer 2

Regulation done by something else other than the gene itself, like a protein binding

Question 3

What are we trying to figure out when studying gene expression?

Answer 3

What sequences are regulating gene expression and what proteins bind to those sequences

Question 4

What techniques will look at the levels or RNA or proteins in a cell?

Answer 4

Western blot, Northern blot, in situ hybridization, RT-PCR, q-RT-PCR, microarray, immunofluorescence

Question 5

What is the conceptually simplest way to determine if a sequence is regulating gene expression?

Answer 5

Mutate it and see what happens

Question 6

Why is it difficult to mutate a suspected regulatory sequence to see what happens?

Answer 6

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

Question 7

Why do we use reporter genes?

Answer 7

Their expression is easy to detect and eliminates the need to design primers, probes, and antibodies for the other gene

Question 8

How are reporter genes used to determine if a sequence of interest is regulatory?

Answer 8

Put the sequence of interest upstream of a reporter gene and detect how expression of the reporter gene changes

Question 9

What is promotor mapping?

Answer 9

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

Question 10

What is enhancer identification?

Answer 10

Placing a sequence of interest very far upstream of a reporter gene to determine if it is acting as an enhancer

Question 11

What is an EMSA?

Answer 11

Electromobility shift assay. Determines if a protein is binding to DNA by looking for a shift created by the bound protein in the gel

Question 12

What does an EMSA allow us to answer?

Answer 12

Does this protein bind to a specific sequence of DNA

Question 13

What creates the shift seen on an EMSA gel?

Answer 13

Protein bound to DNA. It is much larger and will run a lot slower than the unbound DNA

Question 14

What is labelled in an EMSA?

Answer 14

The DNA

Question 15

What is DNase footprinting?

Answer 15

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

Question 16

What does DNase footprinting allow us to answer?

Answer 16

What is the specific DNA sequence that a protein binds to

Question 17

What is labelled in a DNase footprinting assay?

Answer 17

One end of the DNA

Question 18

What is chromatin immunoprecipitation?

Answer 18

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

Question 19

What does chromatin immunoprecipitation allow us to answer?

Answer 19

What are all the sequences in a cell that a protein of interest binds to

Question 20

What is RNA interference?

Answer 20

A regulatory mechanism that occurs in eukaryotic cells at the level of mRNA and translation

Question 21

What is miRNA?

Answer 21

A short piece of RNA that complementary base pairs to mRNA

Question 22

How is miRNA produced?

Answer 22

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

Question 23

What is responsible for processing the long transcript of miRNA into a 70 nucleotide hairpin?

Answer 23

Microprocessor complex

Question 24

What is responsible for processing the 70 nucleotide hairpin into 21 nucleotide duplexes of miRNA?

Answer 24

Dicer endonucleases

Question 25

What is responsible for selecting the correct strand of miRNA that is complementary to the mRNA?

Answer 25

RISC complex

Question 26

What happens to the mRNA when miRNA is perfectly complementary?

Answer 26

It gets rapidly degraded

Question 27

What happens to the mRNA when miRNA has imperfect complementarity?

Answer 27

Translation is blocked

Question 28

Are any miRNAs activators?

Answer 28

No, they only decrease gene expression

Question 29

How common is gene regulation by RNA interference?

Answer 29

Very common, over 50% of human genes are regulated by RNA interference

Question 30

How do researchers use RNAi as a technique?

Answer 30

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

Question 31

Why do we use RNAi as a technique?

Answer 31

It is a great way to shut off gene expression to see what a gene does, and is easier than making mutations

Question 32

What does RNAi allow us to answer?

Answer 32

1. What does this gene do?

2. Where is this gene important