Exam 4: Lecture 8

Question 1

Yeast 1- Hybrid Assay

Answer 1

• designed to identify specific DNA-protein interactions

- used to identify TF’s bound to specific enhancer sequence

Question 2

Yeast-1 Hybrid Assay Step 1

Answer 2

• design transcriptional reporter that contains enhancer element, core promoter, fragment, and reporter like lacZ
• construct integrated into yeast genome

Question 3

Yeast-1 Hybrid Assay Step 2

Answer 3

• clone cDNAs that code for TF’s into plasmids that contain GAL4 activation domain
• each plasmid will contain single cDNA
• when plasmid transcribed and translated will code for chimeric protein that contains GAL4 activation domain fused to individual TF
• entire plasmid library will contain 1000’s of different TF containing plasmids, each fused to GAL4 AD

Question 4

Yeast-1 Hybrid Assay Step 3

Answer 4

• yeast cells containing transcriptional reporter transformed with plasmid library
• each cell plated onto media and allowed to proliferate into colony
• cells treated with analog of lactose
• if TF-AD chimeric protein binds to enhancer it will activate expression of lacZ which will in turn cleave lactose analog

Question 5

Cloning DNA Fragments

Answer 5

• involves digesting genomic DNA with restriction fragments and ligating them into plasmids which can then be transformed into bacterial cells
• genomic DNA and plasmids digested with same restriction enzyme so ends of genomic fragments and plasmid DNA will have same compatible ends
• linear fragments ligated together
• ligated plasmids can be transformed into bacterial cells
• each bacterial cell takes up single plasmid
• plasmid replicated independently of bacterial chromosome and at high levels
• allows for amplification of fragment of interest
• as bacterial cells replicate, form colonies on semi-soft agar medium

Question 6

Cellular components/processes DNA Cloning takes advantage of

Answer 6

• restriction enzymes
• DNA ligase
• plasmids
• antibiotics and antibiotic resistance genes
• bacterial transformation
• DNA replication

Question 7

Plasmids

Answer 7

• naturally occurring cellular pieces of DNA that are found in bacterial and some single-celled eukaryotes
• have own origin of replication (can replicated independently of bacterial chromosome)
• can replicate at higher frequency than bacterial chromosome (could be 1000’s of copies in single cell)
• also carry antibiotic resistance genes that can be transferred during bacterial conjugation

Question 8

Modified Plasmids

Answer 8

• in order to clone and amplify DNA fragments
• still contain origins of replication and antibiotic resistance genes
• contain cluster of restriction enzyme sites called multiple cloning site (MCS).

Question 9

Cloning in Modified Plasmids

Answer 9

• DNA fragments that need to be cloned and amplified placed in MCS
• genomic DNA and plasmid must be digested with same restriction enzyme in order to have compatible or “sticky” ends
• enzymes that leave blunt ends can be used too and are glued into MCS with ligase

Question 10

Role of Antibiotic Resistance Gene

Answer 10

• used as selectable marker to kill bacterial cells that fail to take up plasmid during transformation experiment
• if plasmid contains resistance gene against antibiotic X then the media must be treated with antibiotic X
• any bacterial cells that fail to take up plasmid will be killed by antibiotic
• if antibiotic and antibiotic resistance gene don’t match, then all bacterial cells will be killed regardless of whether or not they have been transformed

Question 11

Yeast-1 Hybrid Test Error

Answer 11

• prone to yield false positives
• second method must be used to confirm potential interactions suggested by Y1H assay
• one method is Electro Mobility Shift Assay (EMSA)

Question 12

Electro Mobility Shift Assay (EMSA) Step 1

Answer 12

• generate radioactively labeled oligonucleotide that contains potential DNA binding site
• usually no longer than ~50 bp and will run to end of agrose gel

Question 13

Electro Mobility Shift Assay (EMSA) Step 2

Answer 13

• purify TF of interest
• then mixed with radioactively labeled oligonucleotide
• mixture treated with chemical that crosslinks protein to DNA

Question 14

Electro Mobility Shift Assay (EMSA) Interpretation of Results

Answer 14

• if TF of interest binds to oligonucleotide then total weight of protein-DNA complex will be larger than oligonucleotide alone and will run higher on gel
• if TF does not bind to oligonucleotide fragment will run at same size as control lane

Question 15

Chromatin Immunoprecipitation (ChIP)

Answer 15

• allow for detection of protein-DNA binding on genome wide scale
• chromatin isolated from cells and digested with restriction enzyme or mechanically sheared
• smaller chromatin fragments are chemically cross-linked to preserve protein-DNA interactions
• chromatin passed through column that contains antibody that recognizes TF of interest
• using 2 different salt concentrations unbound fragments first removed and discarded while bound fragments separated and saved
• chromatin treated with chemical that releases TF from DNA fragments
• fragments can then be sequenced
• any sequence found in common from all fragments likely going to represent binding site of DNA binding protein of interest

Question 16

Why Chromatin Immunoprecipitation?

Answer 16

• can determine binding site for TF

- can determine where in genome TF is bound by looking at sequences that are directly adjacent to TF binding site