Chapter 9

Question 1

How can you study gene function?

Answer 1

1. Molecular and cellular (in vivo) methods. Typically involve using recombinant DNA techniques and insertion of DNA into cells to express gen product in cells.
2. Biochemical (in vitro) methods. Typically involve expression of gene product (protein) in cells. Protein is then purified from cells, and gene function is studied by characterizing the isolated protein (in a reaction outside the cell)

Question 2

in vitro expression system advantages

Answer 2

1. Bacterial = High yield. Highly regulated expression of gene product
2. Yeast = High Yield. More suitable for production of eukaryotic proteins
3. Baculovirus/Insect cells = High yields
4. Chinese hamster ovary (CHO) cells = Can stably integrate expression construct. Modifications (glycans) similar to other mammalian cells

Question 3

in vitro expression system disadvantages

Answer 3

1. Bacterial = Over expression can lead to protein aggregation (insoluble). Incorrect protein folding. Protein can be toxic to cells. Lacks protein modifications that may be necessary for higher eukaryotic proteins
2. Yeast = Glycosylates proteins; however, nature of glycans used is different than mammalian cells
3. Baculovirus/Insect cells = Expression construct is not stable in cells; need to be transfected every time protein expression is desired. Glycosylation differs from mammals
4. Chinese hamster ovary (CHO) cells = Cannot regulate expression as well. More difficult to make copious amounts of protein

Question 4

λPL inducible expression

Answer 4

λ bacteriophage promoter used to regulate expression n bacteria.

1. cI857 encodes for a repressor of transcription that prevents RNA polymerase from initiating transcription of out gene of interest (GOI).
2. Raise temp to 42 C, cI857 unfolds and RNA polymerase can bind to promoter and transcribe GOI

Question 5

T7 bacteriophage inducible expression

Answer 5

System utilizes:
1. T7 bacteriophage RNA polymerase (RNAP) = initiates transcription from T7 promoter
2. T7 lysozyme = enzyme that degrades T7 RNAP
3. T7 promoter upstream of the GOI = recognized y T7 RNAP.
In the absence of IPTG, T7 RNAP is weakly expressed. T7 lysozyme denatures the little T7 RNAP that is present, and the result is no expression of GOI. In the presence of IPTG, T7 RNAP is highly expressed, leading to high expression of GOI

Question 6

General protein purification strategy

Answer 6

1. Induce expression of protein in cells.
2. Lyse cells and recover total protein (whole cell lysate containing poi and other proteins)
3. Pass whole cell lysate through column that retains your poi
4. Elute protein from column

Question 7

Epitope ‘tagging”

Answer 7

Examples = GST and 6x His

Allows for direct purification with little/no empirical determination

Question 8

How nickel charged resin binds to 6x His tag

Answer 8

4 of the Ni2+ ligand binding sits are bound by nitrilotriacetic acid, leaving the 2 remaining ligand binding sites to interact with 2 Histidines of the epitope -tagged protein.
Use imidazole to elute. Know structures

Question 9

Protein characterization in vitro

Answer 9

1. Size
2. Shape
3. Structure
4. Cofactors
5. Monomer or multimer
6. Binding partners
7. Protein/DNA (or RNA) binding
8. Enzyme kinetics
9. Function specific assays

Question 10

Forward (classical) genetics

Answer 10

Studying/identifying the genetic basis for a particular phenotype

Question 11

Reverse genetics

Answer 11

Identifying the phenotype caused by a particular genetic change. Now that DNA can be easily manipulated, once can ask how a particular mutation affects a cell. This allows the cell to tell you the function of the gene product (protein).

Question 12

Tissue/cell culture

Answer 12

To study gene function in mammals, tissue/cell culture is commonly used. Grown in media but take longer. Cell division = 1 day roughly. Require more care

Question 13

Requirements to study gene function in vivo

Answer 13

1. Remove the wild-type gene

2. Replace with a mutant copy

Question 14

Yeast vs higher eukaryotes

Answer 14

1. It is relatively simple to replace genes in yeast through homologous recombination
2. Higher eukaryotes have a low rate of homologous recombination
   Problem: DNA can be integrated into the genome; however integration is random
   Results:
3. Since integration is random, it is very difficult to do exact gene deletion/replacement
4. Gene expression can also be affected by where in the genome the DNA has inserted

Question 15

What is RNA interference?

Answer 15

first discovered in roundworms
Sequence specific silencing of gene expression. Mediated by double stranded RNA - thought to be a defense mechanism from RNA viruses. Highly conserved pathway/machinery. Plays critical roles in gene regulation

Question 16

How does RNA interference work?

Answer 16

Dicing and slicing

1. Double stranded RNA (dsRNA) with complementary sequence to your GOI is introduced into the cell
2. An RNase enzyme called Dicer processes the dsRNA into 20-25 nucleotide small interfering RNA (siRNA).
3. The SiRNA assembles into endoribonuclease containing complexes known as RNA-induced silencing complexes (RISC)
4. siRNA strands guide RISCs to complementary RNA where the RNA is cleaved (and made nonfunctional)

Question 17

Protein localization

Answer 17

Introduce siRNA targeting the mRNA of your GOI and introduce epitope tagged (GFP) constructs into cells. Where the protein localizes is often a good indicator of its cellular function

Question 18

Co-immunoprecipitation

Answer 18

If antibodies are available to your protein of interest, you can determine if the two proteins interact in the cell. Mix protein A and protein B. Add antibody to ProtA. If they interact, ProtB with precipitate with ProtA. If not, you will only get ProtA

Question 19

Yeast two-hybrid assay

Answer 19

2 regions:
1. GAL4 DNA binding domain (GAL4-DBD)
2. GAL4 transcription activation domain (GAL4-AD)
These two regions are necessary to get transcription of a subset of genes or genetically engineered reporter genes. The two domains of GAL4 can be separated and attached to tow different proteins. Even though the two domains of GAL4 have been separated, when they come into close contact transcription of the reporter genes can occur.
Process:
1. Transform GAL4-DBD-YFG1 and GAL4-AD-YFG2 vectors into yeast cells that contain a reporter gene
2. If the two proteins from YFG1 and YFG2 do NOT interact, there will be NO transcription of the reporter genes.
3. If the two protein from YFG1 and YFG2 interact, reporter gene transcription will occur. causing an observable difference (Slide 25)